.'i ^ STUDY IN CEREAL RUSTS: 



PHYSIOLOGICAL RACES 



A THESIS SUBMITTED TO THE FACULTY 

OF 

THE GRADUATE SCHOOL 

OF 

THE UNIVERSITY OF MINNESOTA 

BY 

ELVIN CHARLES STAKMAN 

IN PARTIAL FULFILMENT OF THE REQUIREMENTS 

FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



MINNEAPOLIS, MINN. 
JUNE 1913 



A STUDY IN CEREAL RUSTS: 

PHYSIOLOGICAL RACES 



A THESIS SUBMITTED TO THE FACULTY 

OF 

THE GRADUATE SCHOOL 

OF 

THE UNIVERSITY OF MINNESOTA 

BY 

ELVIN CHARLES STAKMAN 

IN PARTIAL FULFILMENT OF THE REQUIREMENTS 

FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



MINNEAPOLIS, MINN. 
JUNE 1913 



^^ If" 



MAY 12 I»I4 



TABLE OF CONTENTS 

Page 

Part I. Biologic Forms ' 

7 
Historical 

Experimental 

Methods ^^ 

Experim.ents with Puccinia graminis hordei 12 

General statement 12 

Inoculations on rye 12 

Inoculations on oats l-^ 

Inoculation after the use of anesthetics 13 

Inoculations on wheat 1'^ 

Summary of inoculations with Puccinia graminis hordei. . . 15 

Experiments with Puccinia graminis avenae 15 

General statement 1^ 

Inoculations on rye 15 

Inoculations under ordinary conditions 15 

Effect of high fertilization 16 

Effect of anesthetics 16 

Effect of leaf injury 16 

Inoculations on wheat 17 

Inoculations under ordinary conditions 17 

Effect of anesthetics 17 

Effect of manure 18 

Effect of leaf injury .• 18 

Summary of inoculations on wheat 18 

Inoculations on barley 19 

Inoculations under ordinary conditions 19 

Inoculations after exposure to anesthetics 19 

Summary of inoculations made with Puccinia graminis 

19 
avenae ^^ 

Experiments with Puccinia graminis secalis 20 

Inoculations on wheat 20 

Inoculations on oats 20 

Inoculations on barley 20 

Inoculations on einkorn 21 

Experiments with Puccinia graminis tritici 21 

Inoculations on barley 21 

Inoculations on rye 21 

Inoculation under ordinary conditions. 21 



6 CONTENTS 

Inoculation after exposure to ether 21 

Inoculations on oats 21 

Inoculations under ordinary conditions 21 

Inoculation after exposure to ether 22 

Inoculations on oats and rye after the use of barley as a 

"bridging- form" 22 

Summary of experiments with Puccinia graminis tritici. ... 22 

Effect of the aecidial stage on biologic forms 23 

General statement 23 

Experiments in 1912 23 

Experiments in 1913 24 

Adaptation of biologic forms to new hosts 25 

Summary of Part 1 27 

Part II. Rust-resistant varieties of wheat 28 

Historical 28 

Forms which are resistant 29 

Field observations 29 

Experimental 30 

Greenhouse trials 30 

Inoculations on resistant forms 32 

Metabolism of the host and rust resistance 33 

Effect of water content of soil 34 

Effect of fertilizers 36 

The nature of resistance 40 

Histological details of infection 42 

Infection of Minnesota No. 163 42 

Infection of Khapli 44 

The course of infection in other resistant forms 45 

Minnesota No. 163 inoculated with Puccinia graminis avenae 46 

Summary of Part II 48 

Bibliography 50 

Explanation of Plates 55 



A STUDY IN CEREAL RUSTS 
PHYSIOLOGICAL RACES 

PART I. BIOLOGIC FORMS 

HISTORICAL 

Cereal rusts were known as destructive plant pests by the ancients. 
Observations were made on the effect of weather and location on their 
prevalence. The comparative susceptibility of the various cereals 
received some attention. Theophrastus speaks of the varying suscepti- 
bility of different cereals, as does Pliny, who says that barley is less 
likely to rust than are other grains. In modern times many observa- 
tions have been made, but many of these were only incidental. 

Dietel (1887) calls attention to a certain amount of morphological 
and physiological variation in rust fungi, but does not definitely estab- 
lish the fact that there are distinct biologic forms. Previous to his 
time Puccinia graminis Pers. was considered a single species found 
on cereals and various grasses. However, in 1894 Eriksson (1894-1) 
showed that, although the morphology of the fungus on the dift'erent 
cereal hosts varied but slightly, there was a distinct specialization in 
parasitism. He therefore divided the different species of rusts into 
subdivisions which he termed "formae speciales." Puccinia graminis, 
the only species extensively used in the present investigation, he di- 
vided into five formae speciales, two of which he mentioned as being 
sharply demarcated and the remainder as being probably also distinct. 

All of these forms of Puccinia graminis were capable of produc- 
ing aecidia on various species of Berberis. It therefore occurred to 
Eriksson that perhaps his formae speciales would be equalized when 
grown upon the alternate host. This, however, he found not to be the 
case. He concluded that the forms were physiologically distinct even 
when grown upon Berberis. In fact he was led to believe that they 
became more firmly fixed after a period on the barberry. 

This discovery stimulated much research concerning the physiolog- 
ical relationships of the Uredineae. In the United States Hitchcock 
and Carleton (1894) made observations at about the same time that 

7 



8 A STUDY IN CEREAL RUSTS 

Eriksson was making his in Sweden. Their conclusions pointed to 
the fact that there was not much danger of rust from one cereal infect- 
ing another. The same phenomenon was observed in rusts on a wide 
variety of hosts. Magnus (1894 and 1895), Rostrup (1894), Klebahn 
(1896), Dietel (1899), Ward (1901), Bandi (1903), and others firmly 
established the fact that this specialization of parasitism was quite 
common in the various rusts. To the forniae speciales of Eriksson 
various names were applied. Schroeter (see Magnus 1894, p. 360) 
called them Schwester-Arten, and Rostrup (1894. p. 40) called them 
biologische Arten, while Hitchcock and Carleton (1894) referred to 
them as physiological races. 

Neger (1902) found evidences of a similar condition among the 
Ervsiphace?e. Marchal (1902) conducted a large number of cross-inoc- 
ulations with Erysiphe graminis and concluded that there was a fairly 
large number of "races specialisees." These did not differ essentially 
in any morphological character. He showed (1903) and Salmon 
(1903-2) later showed that the ascospores behaved in the same way 
with regard to this specialization of parasitism as did the conidia. 
Reed (1905) has shown that there may be more than one physiological 
race upon a single genus of host plant. 

Magnus was one of the first to try to explain the phenomenon of 
specialization of parasitism. He distinguishes (1894, p. 366) be- 
tween "Gewohnheitsrassen" or adaptive races and biologic forms. 
The former name he applies to such forms as merely show difference 
in infection power, while those which are fixed he calls biologic forms. 
Long association with one host plant, he says, may bring the develop- 
ment from Gewohnheitsrassen to biologic forms. This he showed 
(1895) to be true not only of rust fungi but of others as well. Dietel 
(1899) expressed a somewhat similar opinion, his idea being that 
formerly a given species attacked a variety of hosts, but that it be- 
came more and more specialized to form first Gewohnheitsrassen and 
then biologic forms. Eriksson (1902, p. 657) says that rust forms adapt 
themselves. Where a certain host is present in large numbers, and 
climatic conditions are favorable, changes take place in favor of the 
new host. These changes are expressed not only in the vitality of the 
fungus but also in a higher degree of systematic firmness. The new 
rust form, he says, becomes separated from its sister forms of parallel 
•origin and becomes "scharf fixiert." His conclusion is, "Das Phanomen 
der Spezialisierung steht nicht langer da als der Exponent eines dem 
Schmarotzer innewohnenden, launenhaften und unerklarlichen Triebes, 
iieue Formen zu produzieren. Dieser Trieb wird durch die umge- 
benden Verhaltnisse — die vegetative Unterlage und das Klima, — 
unter denen der Parasit lebt, in eine bestimmte Richtung geleitet." 
This, Eriksson (1902, pp. 606 and 654) thinks, accounts for the fact 



BIOLOGIC FORMS 9 

that the specialization has taken a different course in Sweden from 
that it has followed in the United States. The most widely grown 
crops would naturally be the ones on which the particular biologic 
forms adapted to them would attain their highest development. There- 
fore, the fact that a rust shows particular relationships in one country 
does not by any means preclude the possibility of a quite different set 
of relationships in another country. 

The idea that the fungus changes its habits as a result of environ- 
ment is substantiated by many observations. Perhaps an extreme case 
of such a tendency is found in the life history of Puccinia graminis in 
Australia. McAlpine (1906, p. 21) states that the teleutospores seem 
unable to infect the barberry in Australia, and, according to his obser- 
\ations, the fungus is quite rapidly being reduced to a reproduction 
by uredospores only. This is accounted for by the absence of the bar- 
berry. 

Eriksson (1896, p. 339) shows that closely related host forms 
are somewhat similar in their relation to rust. He says, however, that 
the taxonomic relationship of host plants does not entirely determine 
the specialization of the rust form. Ward (1901) in his work on the 
rust (Puccinia dispersa) of the bromes states that the closeness of re- 
lationship of hosts is the determining factor in the ability of the rust 
to pass successfully from one host plant to another. Freeman (1902) 
also concluded that the farther removed a species of Bromus was tax- 
onomically from the plant serving as a host for the rust the less prob- 
ability there was of infection. Ward showed further (1903j that some 
forms of bromes might act as bridging species in enabling the rust to 
pass indirectly from one group of bromes to another, although direct 
transfer was impossible. Salmon (1904) showed that the same thing 
was true of Erysiphe graminis D. C. Freeman and Johnson (1911) 
have found that barley can act as a bridging form enabling Puccinia 
graminis to increase its range of infection power. Salmon (1904 and 
1905) sl:owed that the range of infection possibility of Erysiphe 
graminis forms may also be increased under certain cultural conditions. 
By injuring leaves and subjecting plants to heat and anesthetics he 
was able to infect normally immune forms. 

The conception of a biologic form, then, is that it represents a 
tendency toward adaptation. This tendency may be due to various 
causes, the evidence being that it depends largely on the availability 
of host species. Hitchcock and Carleton (1894), Carleton (1899), and 
Freeman and Johnson (1911) investigated quite thoroughly the mat- 
ter of biologic forms of Puccinia graminis in the United States. Free- 
man and Johnson (1911, p. 27) give the following as the biologic 
forms of this rust in the United States : 

P. graminis tritici (stem rust of wheat) on wheat and barlev. 



10 A STUDY IN CEREAL RUSTS 

P. graminis hordei (stem rust of barley) on barley, wheat, and 
rye. 

P. graminis secalis (stem rust of rye) on rye and barley. 

P. graminis avenae (stem rust of oats) on oats. 

If these forms are merely adaptations it ought to be possible to 
change their parasitic tendencies by restricting or changing their en- 
vironment. It ought to be possible to break down the biologic forms 
under conditions abnormal for host or parasite. Various methods 
have been tried in attempting to break down the specialization of para- 
sitism of different fungi. Salmon's work along this line on the Ery- 
siphaceae has already been mentioned. Ray (1903) states that by 
subjecting maize to ether vapor and then inoculating it with spores of 
Ustilago zeae, the resulting infection was much more virulent than 
that on plants not so treated. 

Comparatively little work of this nature has been done with Puc- 
ciiiia graminis. The fact that physiological races of rusts behave dif- 
ferently under different conditions has been known for some time. 
Much of the information was, however, gathered from incidental 
observations. In breeding wheats for the purpose of obtaining rust- 
resistant forms it would be very helpful to be able to correlate certain 
characters with rust resistance. For this reason this phase of the 
question, from both practical and scientific points of view, is of much 
importance. The same is true of physiological races. It is important 
to know if they can be broken down by means of a high degree of soil- 
fertilization, if they become generalized by growing on the alternate 
host, and if they adapt themselves readily to new hosts. The present 
investigation was therefore undertaken with the object of determining 
the possibility of developing and breaking down physiological races 
and of obtaining definite information concerning the factors influencing 
varying resistance in immune or semi-immune varieties of wheat. 

EXPERIMENTAL 

Methods 

The rusts used in making inoculations were obtained originally 
from their respective hosts in the fields at University Farm, St. Paul, 
Minnesota. They were then artificially transferred to plants growing 
in the greenhouse. Transfers were made to new plants about once 
every three weeks until the rust had been confined to its own host for 
at least twelve successive transfer generations. In nearly all the exper- 
iments with biologic forms the rust had been confined to its own host 
for at least twenty generations, thus giving assurance that it was the 
particular biologic form desired. 

The seeds of the host plants were planted in rich loam soil in four- 



BIOLOGIC FORMS 11 

inch clay pots. Only ten plants were left in each pot and the first leaf 
of each was inoculated when six or seven days old. The plants were 
trimmed whenever necessary so as to leave only the one inoculated leaf 
on each plant. Fresh, viable uredospores were used for inoculations 
except where otherwise specified. The spores were put on the leaves 
with a flat inoculating needle which had been previously moistened 
in order that the spores might better adhere to the leaf surface. The 
pots were then placed in shallow pans filled with water, or on wet 
sand, and covered with bell jars for forty-eight hours. In nearly all 
cases a fine film of moisture covered the leaves during a considerable 
part of the time that they were under the jars. This, together with a 
moderate temperature, made the conditions for infection ideal. After 
the removal of the bell jars the plants were kept on greenhouse benches 
in such locations as to reduce to the minimum the danger of accidental 
infection. 

The grains used were the following, the numbers, except where 
specified, being Grain Investigation numbers of the United States 
Department of Agriculture: 

Fife wheat, Minnesota No. 163 

Velvet Blue Stem wheat, Minnesota No. 169 

Minnesota No. 188 wheat, a cross between White Fife and Ladoga 
Fife 

Manchuria barley, Minnesota No. 105 

Early Gothland oats, Minnesota No. 295 

Swedish rye, Minnesota No. 2 * 

Kubanka 1516 — Nos. 8 and 9 pedigreed — Dickinson, N. Dak., 1910 

Kubanka 2094 

Arnautka 288 

Arnautka 1431 

lumillo 1736 (1736-II-3 selected at Amarillo, Tex., 1910) 

Einkorn (Triticum monococcum) 2433 — Nos. 4, 6, 7, and 8 pedi- 
greed — Dickinson, N. Dak., 1910 

Emmer (Triticum dicoccum) 1522 

Khapli (an Indian Emmer) 

Of these the Kubankas, Arnautka, and lumillo are varieties of 
Triticum durum. The durums generally have the reputation of being 
more resistant to rusts than are the ordinary wheats (Carleton 1905, p. 
9). Einkorn, also, is quite resistant while the emmers vary greatly, 
one used in this work, G. I. No. 1522, not being very resistant. Khapli 
is an emmer obtained from India by E. C. Johnson, formerly Cereal 
Pathologist in the office of Grain Investigations, United States Depart- 
ment of Agriculture. It has no Grain Investigation number. It is the 
most resistant of all the forms used. 



12 A STUDY IN CEREAL RUSTS 

Experiments with Puccinia graminis hordei 

general statement 
Freeman and Johnson (1911, p. 20) state that the barley stem 
rust is more versatile than any of the other biologic forms of the cereal 
rusts, and, further, that the range of infection of a given form is in- 
creased after having been transferred to barley. The results of the 
author, in general, agree with those of Freeman and Johnson, although 
the percentages of infection are in some cases quite different. 

inoculations on RYE 

In the first series 20 leaves were inoculated, after which the pots 
were kept under bell jars for 48 hours. In 8 days very distinct signs 
of infection began to appear and on the tenth day distinct pustules 
were quite numerous. Eventually 16 leaves out of 18 showed distinct 
pustules. Two of the leaves had been killed. Very evidently not all 
of the mycelial wefts developed pustules, since pustules were often 
intermingled with typical, yellowish rust flecks and green islands. The 
pustules were in all cases small, most of them less than a millimeter in 
size, but they were fairly numerous. There were some fairly large 
areas in which no pustules developed, but which contained a large 
number of green islands. 

In the second series 40 leaves were inoculated, 10 of which eventu- 
ally produced pustules. Of the remaining 30 leaves 29 were very 
strongly flecked in such a manner as to show conclusively that infec- 
tion had taken place although no pustules had been formed. 

The results of both series indicate that rye is easily infected by 
the stem rust from barley. Out of 58 leaves, 26 were infected, indi- 
cating greater ease of infection than in case of the strain used by 
Freeman and Johnson (1911, p. 19) in their experiments. The 29 
leaves which are indicated as having been strongly flecked would at 
first sight have been counted as having pustules. However, close 
examination showed that there were no ruptured pustules. 

Although a fairly high percentage of successful infections can be 
obtained, rye is by no means a congenial host for Puccinia graminis 
from barley. This is shown by the fact that the pustules are always 
small. Long areas of the leaf may be killed and in this area there may 
be many unruptured pustules. Often an area extending across the 
entire leaf and one centimeter or more in length may be completely 
killed. (See Plate I, A.) This may not contain a single pustule, but 
close examination reveals the fact that there are many green islands, 
some of which have a yellowish tinge in the center. These latter look 
very much like unruptured pustules. Histological examination re- 
veals the fact that the mycelium has spread, the host cells have died, 
and wefts of mycelium have formed, either directly under, or a slight 



BIOLOGIC FORMS 13 

distance below, the epiderm. These wefts may send up a few hyphae 
which resemble those normally producing spores. In some cases a 
few small, abortive spores are formed, while in other cases, none are 
produced. 

This phenomenon very closely parallels that occurring in some of 
the resistant forms of wheat. Attention will be called to this more in 
detail later on. It will be sufficient here to emphasize the fact that 
rye, when inoculated with Puccinia graminis spores from rye, does not 
show any of these dead areas (see Plate I, B), the leaves remaining 
green and producing pustules vigorously. When the spores, however, 
are taken from barley, infection takes place, but there is not such a per- 
fect relationship set up between host and parasite as to enable both to 
live and thrive for a long time. 

INOCULATIONS ON OATS 

The first attempt to infect oats with stem rust from barley failed 
absolutely. For this reason the greatest precautions were taken to 
furnish optimum conditions for germination of spores and develop- 
ment of the mycelium. Of 104 leaves inoculated at various times, not 
one produced pustules. Of this number 8 were slightly flecked, but 
showed no tendency to form pustules. In fact, the flecks were ex- 
tremely small and very few occurred on each leaf. In some cases 
there was but a single fleck. These might quite easily have escaped 
detection on some of the leaves had they not been invariably situated 
in the inoculated area. Their position in this area indicates quite 
strongly that they were true rust flecks. In these areas the tissues of 
the leaves seemed to have been killed outright. However, the mycelium 
must have been very restricted in its development since the diameter 
of a spot never exceeded one millimeter. 

Inoculation after the use of anesthetics — 

In the first trial two pots of oats were exposed to the fumes of 
ether for 15 minutes. Immediately after the exposure they were 
inoculated with fresh spores of Puccinia graminis from barley and 
then placed under a bell jar in a shallow pan of water. Two pots 
were exposed to chloroform fumes for an equal length of time and 
inoculated. At the same time 25 leaves were inoculated under normal 
conditions, as a check. 

Of those leaves exposed to ether, 4 out of a total of 20 developed 
slight flecks but no pustules ; neither were there any unruptured pus- 
tules, which are commonly found when infection is not normal. Of 
those exposed to chloroform 2 out of 19 developed pustules in 12 
days. These pustules were very small, but were unquestionably pus- 
tules of stem rust. A few of the leaves were so indistinctly flecked 
that it was doubtful if they were true rust flecks. 



14 A STUDY IN CEREAL RUSTS 

111 the second trial plants were again exposed to ether and chloro- 
form, but the time was increased from 15 to 20 minutes. An attempt 
was made to insure conditions for successful infection. It was ob- 
served that a film of moisture formed on the leaves at night and the 
temperature was moderate, insuring successful infection if that was 
ordinarily possible. 

Of the 20 leaves exposed to ether, 11 became very distinctly 
flecked, but there were no evidences whatever of successful pustule 
formation. In the chloroform series one leaf out of 20 produced a 
small pustule and 8 became clearly flecked. There were 20 check 
leaves, and, of these, 4 became flecked. The flecking was, however, 
rather indistinct. 

Out of a total of 193 inoculations only 3 resulted in pustule-for- 
mation, and these only after exposure to chloroform. Of the remain- 
der, 31 were flecked, but this flecking was not always sharp on the 
control plants. In the other cases, however, it was distinct. The spots 
were always less than one millimeter in diameter, and usually much 
smaller. It appeared that this small area of the leaf had been killed, 
thus preventing the further spread of the mycelium and precluding 
the possibility of pustule formation. In no case was there any indica- 
tion whatever that large areas of the leaf were involved, as was the 
case when rye was inoculated with the rust from barley. In this con- 
nection it may be mentioned that Freeman and Johnson (1911, p. 19) 
were able to get 7 successful infections out of a total of 35, but the 
pustules were always very small. It may be that the difference is due 
to the use of various strains of the rust. The fact that there are strains 
of the same biologic form seems to be quite definitely indicated. In 
any case this seems to furnish an example of rather extreme incom- 
patibility between host and parasite. In some cases it would seem that 
the infection threads of the fungus are checked almost immediately; 
or that they may gain a temporary foothold, only to kill the cells upon 
which they depend for nourishment and then develop but little further. 

INOCULATIONS ON WHEAT 

There is no question but that the stem rust from barley, in this 
country, usually passes to wheat with practically the same degree of 
readiness with which it passes to barley. In the trials made with this 
form this appeared to be the case whether susceptible or resistant forms 
of wheat were used. Inoculations on Arnautka, Khapli, and emmer 
show that the barley rust is quite as capable as is the wheat rust of 
attacking these varieties. 

Pritchard (1911-1, pp. 181, 182) cites evidence tending to show 
that the forms on wheat and barley in North Dakota are distinct. This 
is not the case, however, with the strains used in the experiments in 
Minnesota. 



BIOLOGIC FORMS IS 

SUMMARY OF INOCULATIONS WITH PUCCINIA GRAMINIS HORDEI 

Wheat and barley are not included since in nearly every case IOC 
per cent of infection results. The denominator in each case represents 
rhe total number of leaves inoculated, and the numerator, the number 
of leaves which developed pustules. 
Barley rust to : 

OatSy^^; 8 slightly flecked 

Oats after exposure to ether ,o . j^ flecked 

Oats after exposure to chloroform -g^^g ; 8 flecked 
Rye-|-^; 29 very strongly flecked 

It will thus be seen that barley rust -does not find either rye or oats 
a congenial host. It was transferred to rye much more easily in these 
experiments than in those reported by other investigators in this coun- 
try. Without exposing the host plants to anesthetics, however, no suc- 
cessful infection of oats was obtained. By the use of ether and chloro- 
form the possibility of infection was somewhat increased, as evidenced 
by the formation of pustules on a small percentage of leaves and the 
increased percentage of flecked leaves. In addition to this, the flecks 
were much more distinct than those on the check plants. 

Experiments with Puccinia graminis avenae 
general statement 
This rust in this country is supposed to infect no cereal except 
oats, although it is capable of infecting a number of grasses. Carleton 
(1899, p. 60) was unable to transfer it successfully to wheat, barley, 
or rye. Eriksson (1902, p. 601) mentions it as being found on oats 
and 18 species of grasses in Sweden. Freeman and Johnson (1911, 
p. 22), however, find that it can be transferred to barley also, and they 
report that Derr succeeded in obtaining direct transfers from oats to 
wheat and rye. 

INOCULATIONS ON RYE 

Since Derr, according to Freeman and Johnson (1911, p. 22), 
obtained but one successful infection by inoculating rye with stem 
rust of oats, a large number of trials were made with this form, espe- 
cially since the same authors (I.e. p. 23) assert that under favorable 
conditions these can undoubtedly be made. 

Inoculations under ordinary conditions — 

The results of inoculations made imder average conditions show 
clearly that it is possible to transfer the rust from oats to rye. Al- 
though the percentage of entirely successful infections was small, the 



16 A STUDY IN CEREAL RUSTS 

appearance of inoculated plants would lead one to believe that the rust 
transfers with greater ease than it really does. Out of a total of 55 
leaves inoculated only 3 produced pustules. These pustules, althougii 
small, were very distinct. Of the remaining leaves, 13 were clearly 
flecked. It is the appearance of these so-called flecked leaves which is 
often misleading. When looking at them from a distance they some- 
times appear to be quite badly rusted. Closer examination, however, 
sliows that there are no pustules. Sometimes the flecks are so distinct 
as to suggest the appearance of unruptured pustules. 

The effect of high fertilisation — 

A rich loam was well mixed with rich barnyard manure. Rye 
was planted in two pots and the leaves inoculated in the usual manner. 
Evidences of infection appeared at the usual time. It was found thai 
the severity of infection was much greater than that on plants grown 
in unfertilized soil. Of a total of 18 leaves 9 developed pustules and 
the other 9 were distinctly flecked. The areas of infection on these 
pknts were much larger than on those grown in ordinary soil. The 
leaf tissues were very clearly killed, sometimes in areas a centimeter 
long, and in these areas small pustules appeared. The appearance 
was very characteristic of semi-normal infection. The mycelium spread 
fairly well, but the host cells were killed and o,nly small pustules were 
developed. 

The effect of anesthetics — 

Plants were inoculated after exposure to ether, chloroform, and 
nitrous oxide for periods ranging in different experiments from 5 to 
15 minutes. The difference between the check plants and those ex- 
posed to anesthetics for 10 minutes was fairly distinct. This was not 
apparent so much in the number of pustules or flecks, but rather in the 
sharpness of the flecks. The following were the results : 

After exposure to ether -g'-^-; 26 flecked 
After exposure to chloroform -^-^\ 13 flecked 
After exposure to nitrous oxide ^^-5-; 6 flecked 

It will thus be observed that in point of numbers the difference 
between the success of these infections and those on the check plants 
was not great. However, a real difference in severity of infection, 
although not remarkable, did exist. On the other hand the plants in 
highly fertilized soil developed a larger number of pustules and also a 
more typical infection. 

Effect of leaf injury — 

A number of leaves were injured, just previous to inoculation, by 
being punctured in many places with a sterilized needle point. Spores 
were then placed on this injured area in large numbers. The epidermis 



BIOLOGIC FORMS 17 

was stripped from others and the inoculations then made. The results 
were not different from those obtained on check plants. Only one 
pustule developed on one leaf out of a total of 28. Six of the remain- • 
ing leaves were flecked, but the flecks remained small. In no case did 
the virulence of infection equal that on plants grown in highly fertilized 
soil. 

The stem rust of oats, then, can be transferred directly to rye. 
This was accomplished most easily when the plants were grown in 
heavily manured soil. Exposing them to various anesthetics before 
inoculation seems to increase the virulence of infection slightly, while 
leaf injury had no apparent effect. In the various trials, under differ- 
ent conditions, 27 out of 236 leaves developed pustules and 73 were 
flecked. Under conditions which might exist in the field 12 out of 73 
leaves produced pustules and 22 were flecked. 

INOCULATIONS ON WHEAT 

The stem rust of oats can be transferred to wheat only with great 
difficulty. Carleton (1899, p. 60) did not succeed in obtaining infec- 
tion in his experiments. Freeman and Johnson (1911, p. 22) made 
100 inoculations but none was successful. They report, however, that 
Derr was able to make transfers. 

Inoculations under ordinary conditions — 

The fact that the oat rust is transferred with great difficulty to 
wheat became apparent very soon. Out of 108 inoculations only one 
was entirely successful, although 5 leaves became slightly flecked. 
Very evidently the infection threads, after having grown among the 
tissues of the leaf, must have died, since only very minute flecks de- 
veloped, and these in only a few cases. 

Effect of anesthetics — 

Plants were inoculated after exposure to ether and chloroform 
for periods of from 1 to 15 minutes. The best results were obtained 
after exposure to ether for 5 minutes. In this case 3 leaves out of 50 
developed pustules while all the other leaves were flecked. These 
pustules and flecks appeared 10 days after inoculation, this being 
slightly longer than a normal incubation period. None of the check 
plants showed any distinct signs of successful infection, while the 
flecks on the plants which had been exposed to ether were very con- 
spicuous, forming a sharp contrast with the check plants. These 
flecks were scattered all along the line of inoculation and had the 
appearance of young, unruptured pustules. 

This series offered the best evidence that anesthetics may have 
some influence in rendering a plant more susceptible to a rust than it 
would otherwise be. In subsequent trials, exposing the plants for a 



18 A STUDY IN CEREAL RUSTS 

greater length of time, no pustules were developed on any of the 40 
leaves inoculated. Nine of these leaves were, however, quite strongly 
flecked. The use of chloroform did not result in the formation of 
pustules, although there was a distinct advantage over the check plants 
both in number and definiteness of flecks. In all, 40 plants were inocu- 
lated and of these 19 became distinctly flecked. After the use of ether, 
then, 3 out of 90 leaves were successfully infected, and 56 were sharply 
flecked, and these, added to the totals after the use of chloroform, give 
3 pustules and 75 infected leaves which developed no pustules out of 
130 trials. 

Effect of manure — 

Wheat was planted in two pots containing a rich loam very heavily 
fertilized with rich barnyard manure. Of the 20 leaves inoculated, 6 
developed pustules and the rest were strongly flecked. In this case, 
however, there was a possibility that the pustules developed as a result 
of accidental infection. The flecks were apparently due to the arti- 
ficial inoculation, since such flecks have never been observed after 
direct inoculation with spores from either wheat or barley, or after 
the inoculation of wheat with wheat or barley rust. It is therefore 
quite certain that the pustles, which were normal, were the result of 
accidental infection, while the flecks, which were exactly like those 
developed on a semi-immune form, were the result of artificial inocula- 
tion. 

Effect of leaf injury — 

In one experiment, 16 leaves were pricked full of holes in an 
area of one centimeter or more. They were then inoculated, and 4 
became flecked, but no pustules developed. In another experiment the 
epiderm was stripped from 29 leaves immediately before inoculation. 
Although 10 became flecked, the flecks were extremely minute and no 
pustules were developed. Histological examination showed that the 
spores had sent out germ tubes in large numbers. These tubes grew 
among the host cells, but true infection did not take place. Sections 
of these plants were made and examined. It was clearly evident that 
leaf injury did not increase the chances for infection. The hyphae did 
not develop better than did those in normally inoculated plants. 

Summary of inoculations on zuheat — 

Out of a total number of 283 leaves inoculated under varying 
conditions only 4 developed pustules and 113 became flecked, showing 
that, although the rust of oats can sometimes develop on wheat, it can 
seldom attain to pustule formation. The severity of infection, always 
very slight, can be increased somewhat by exposing plants to be in- 
oculated to anesthetics. The experiment with high fertilization of 



BIOLOGIC FORMS 19 

soil seems to indicate that here, as well as in the case of rye, inoculated 
with oat rust, the mycelium is enabled to develop more extensively 
and possibly produce more spores than when ordinary soil is used. 

INOCULATIONS ON BARLEY 

The results of this series of inoculations were very similiar to 
those obtained by other investigators. The behavior of the rust was 
typical of its usual behavior on an uncongenial host plant. The flecks 
were small in nearly all cases and were especially sharp when plants 
had been exposed to anesthetics. 

Inoculations made under ordinary conditions — 

These were not especially successful. No pustules were developed 
on 50 inoculated leaves but 10 of the leaves became flecked. 

Inoculations after exposure to anesthetics — 

Ether and chloroform were used. The period of exposure varied 
at diflferent times from 5 to 15 minutes. Plants exposed only 5 minutes 
did not appear different from the check plants. After exposure for 
15 minutes the flecking was more distinct than on the check plants. 
Only one pustule developed in the entire number of trials, this being 
after exposure to ether. There were 47 leaves inoculated after expo- 
sure to ether, and of these one developed a pustule and 15 were fleck- 
ed. The flecks in this case had every appearance of unruptured pus- 
tules. No pustules were developed after exposure to chloroform, 
but 7 leaves out of 50 became flecked. Of 147 leaves inoculated 32 
were flecked, but only one showed a pustule. 

Summary of Inoculations made with Puccinia graminis avenae 
Oat rust to: 

Wheat ^^^- : 5 flecked 

Wheat after exposure to ether ^j^^ ; 56 flecked 

Wheat after exposure to chloroform ^ ; 19 flecked 

Wheat after leaf injury^; 14 flecked 

Wheat plants grown in manured soil -^-^ ? 

Barley -^^ ; 10 flecked 

Barley after exposure to ether -^y ; IS flecked 

Barley after exposure to chloroform -^ ; 7 flecked 

Rye -|^; 13 flecked 

Rye after exposure to ether -g^'-^; 26 flecked 

Rye after exposure to chloroform -j^^; 13 flecked 

Rye after exposure to nitrous oxide ^.; 6 flecked 

Rye after leaf injury ^^; 14 flecked 

Rye grown in highly fertilized soil -^^ ; 9 flecked 



20 A STUDY IN CEREAL RUSTS 

The rust can be transferred successfully from oats to any one of 
the other cereals. It infects rye much more easily than wheat or bar- 
ley. Anesthetics help to break down the barriers as does a high degree 
of soil fertilization. It should be mentioned that this fact seems due, 
not to any new ability the rust fungus has of attacking an uncongenial 
host, but to an increased capacity for development. Here again evi- 
dence of the possible nature of resistance is ofTered by the inoculated 
plants. The flecks, whether large or small, consist in many cases of 
dead tissue of the host plant. Histological examination shows that the 
fungus gains entrance but cannot develop to any extent in these areas 
which are killed by the fungus itself. 

Experiments with Puccinia graminis secalis 
These experiments were not very extensive and were made with 
the idea of determining rather the character than the number of suc- 
cessful infections. 

inoculations on wheat 
In all 70 leaves were inoculated. None of them produced pustules, 
but 18 became flecked. The flecks were distinct but in no case were 
they large. There seemed to be much less dead tissue than is the case 
in many of the other forms, as for instance rye inoculated with barley 
rust. In fact the flecks were hardly noticeable unless one looked very 
carefully for them. Apparently they were due to the death of host- 
plant cells in the inoculated area. 

inoculations on oats 
In addition to a small number of control inoculations, some were 
made after exposure to ether and others after exposure to chloroform. 
The anesthetics apparently made no particular di:fference in the suc- 
cess of the attempts, although there were more flecked leaves after ex- 
posure to chloroform. Nothing out of the ordinary appeared so only 
the summaries are given : 

After chloroform, 5 min. y*^; 5 slightly flecked 

After ether, 5 min. -^ ; 2 slightly flecked 

Under ordinary conditions -g^; 1 slightly flecked 

INOCULATIONS ON BARLEY 

After exposure to ether for 5 minutes, 16 out of 21 inoculated 
leaves became very distinctly flecked. There were many of these 
flecks all along the line of inoculation. They were very suggestive of 
the flecking in other forms in which the mycelium was known to have 
spread to some extent. However, no pustules were developed. Judg- 
ing from the experience with other forms, if these plants had been 
highly fertilized they might have developed pustules. Of the check 



BIOLOGIC FORMS 21 

plants, only 4 out of 40 became flecked. No pustules were developed. 

INOCULATIONS ON EINKORN 

Einkorn was inoculated with rye rust both after exposure to 
ether for 15 minutes and without having been so exposed. The length 
of time in ether fumes was 15 minutes. No pustules developed on 
any of the leaves, 20 being used in each series. 

Experiments with Puccinia graminis tritici 
inoculations on barley 
It has long been a well-established fact that stem rust from wheat 
can easily attack barley. The infection in trials made by the writer 
was found to be normal and practically as virulent as if spores had 
been taken directly from barley. In the one series tried especially for 
the purpose 30 out of 30 leaves became characteristically infected. 
There is no evidence whatever of uncongeniality between host and 
parasite. 

INOCULATIONS ON RYE 

Inoculation under ordinary conditions — 

In the control experiments 6 out of 30 leaves developed pustules 
and 20 were strongly flecked. In fact, they were so strongly flecked 
that flecking hardly expresses properly the appearance developed. 
Long areas on the leaf were killed outright. In these dead areas very 
small green islands were often found, some of which contained un- 
ruptured pustules. All the pustules were very minute, and some had 
ruptured the epiderm. It was another very good example of semi- 
compatibility between host and fungus. The host leaves did not suffer 
very great injury; the fungus was enabled to spread to a certain ex- 
tent but succeeded in producing only small pustules. 

Inoculation after exposure to ether for five minutes — 

The results here were very striking. Fifty leaves were inoculated, 
6 of which produced pustules, while every leaf was infected with the 
mycelium of the rust fungus. The character of the infection was much 
the same as was that on the check plants. The areas were perhaps 
more extended. On some leaves there were dead areas 3 centimeters 
long, while ordinarily they were not so long on the check plants. 

INOCULATIONS ON OATS 

Inoculations under ordinary conditions — 

Direct inoculation of oats by spores from wheat has not met with 
success on the part of either Carleton (1899, p. 54), who, however, 
reports a doubtful case, or Freeman and Johnson (1911, p. 18), who 
cite Derr as authority for the statement that this direct transfer can 
be made. 



22 A STUDY /.V CEREAL RUSTS 

Attempts to transfer directly from wheat were unsuccessful. Not 
a large number were made, but of the 46 attempts no leaves produced 
pustules and only 2 of them developed flecks. The flecks were very 
small and not very distinct. In this connection it may be mentioned 
that when oat leaves were inoculated with aecidiospores derived from 
wheat teleutospores one leaf out of 56 developed pustules. This ap- 
pears to indicate that direct transfer can be made. The success of the 
inoculation was probably not due to the fact that the rust had passed 
through the barberry stage. This seems especially true since the 
other cereals behaved in the same way toward the aecidiospores as 
they did toward the corresponding uredospores. 

Inoculation after exposure to ether — 

Onlv 30 leaves were inoculated after having been in ether fumes 
for from 3 to 5 minutes. Xo pustules were developed, but 10 of the 
leaves were flecked. There was a perceptible difference between the 
check plants and those of this series, although it cannot be said that 
the results were very striking. 

INOCULATIONS ON 0.\TS AND RYE AFTER THE USE OF BARLEY AS A 

BRIDGING FORM 

The work done on bridging species has already been mentioned. 
Since it is almost impossible to infect oats directly with wheat aecidio- 
spores or uredospores, attempt was made to transfer aecidiospores de- 
veloped from wheat teleutospores first to barley and then to infect 
oats with the resulting uredospores. The attempt was made with 
fourth- and fifth-generation uredospores from barley, these uredo- 
spores having been derived from wheat-rust aecidiospores. Successful 
infection took place in one out of 39 attempts, 4 leaves being distinctly 
flecked. 

The rve plants are possibly slightly more severely attacked if the 
rust is transferred first to barley, 16 out of 19 of the plants inoculated 
becoming infected. 

Summary of Experiments with Puccixi.\ gr.^minis XRiTia 
Wheat rust to: 

Barley II 

Rye ^; 20 flecked 

Rye after exposure to ether -J^ ; 43 flecked 

Rye after barberry and 4 generations on barley -j-| 

Oats, uredospores -f^; 2 flecked 

Oats, aecidiospores -^ 

Oats after exposure to ether -^^ ; 10 flecked 

Oats after barberry and 4 generations on barley ^; 4 flecked 



BIOLOGIC FORMS 



23 



El-'FECT OF THE AeCIDIAL StAGE ON BlOLOGIC FORMS 
GENERAL STATEMENT 

Eriksson (1894, pp. 292-309) was one of the first to suggest the 
possibihty of breaking down biologic forms by means of the aecidial 
generation. He came to the conclusion, however, that this could not 
be done, that, as far as the cereal rusts are concerned, they behave in 
exactly the same manner when transferred through barberry as they 
do in the urediuial stage. Salmon (1903, p. 159) and Marchal (1903, 
p. 280) showed that with biologic forms of the Erysiphaceae the rela- 
tions were exactly the same whether ascospores or conidia were used. 
There is not an exact parallelism between the two since the cereal 
rusts are heteroecious. However, the cases are somewhat similar since 
a sexual fusion intervenes in both. Arthur (1910, pp. 227-228) con- 
cludes that although there is distinct specialization of parasitism in 
Puccinia poculiformis (Jacq.) Wettst. (Puccinia graminis Pers.) on 
various grasses, this specialization breaks down in the aecidial stage. 
The barberry would, then, serve as a bridging form between the vari- 
ous grasses. Jaczewski (1910, pp. 356-357), on the other hand, does 
not find this to be the case with biologic forms of Puccinia graminis 
on cereals and grasses in Russia. His experiments support Eriksson's 
claim that the barberry does not change the physiological specialization 
of the various strains when they produce aecidia. 

EXPERIMENTS IN 1912 

In connection with the experiments described by the writer it 
should be mentioned that the aecidia used in one set of experiments 
were developed in the field but there was very slight chance for acci- 
dental infection. Wheat straw very badly aflfected with rust in the 
teleutospore stage was tied around barberry bushes. There was no 
other rusted straw of any kind within considerable distance, so that 
there was little chance of accidental infection. These aecidiospores 
which were developed from wheat rust were transferred to wheat, 
barley, oats, rye, and einkorn. The following were the results : 

Results of Inoculations of Cereals with AEcioiosroREs of Puccinia graminis 
TRiTici Developed in the Field 



■ 

Grain 


Trials 


Total 


I 


II 


Wheat 


2 1 

3 1 


2 9 
■SIT 


5 
61 


Barley 


1 2 


2 3 


3 5 


Oats 





1' 


1 
6 6 


Rye 


4 


1 1 

■2-S" 


1 5 
"FT 


Einkorn 


1 8 


2 4 
"3U 


4 2 
'611 



•Somewhat doubtful. 



24 



A STUDY IN CEREAL RUSTS 



It will be noticed that the percentages of infection are practically 
the same as are those developed from uredospore inoculation. This 
is true also of the character of the infection. 

On the rye plants, for instance, there was the same characteristic 
spotting and the same small, abortive pustules. The pustules on rye 
were all very small and there was no observable increase in virulence. 
The same thing is true of einkorn. On oats but one rather doubtful 
pustule developed, indicating that the aecidial stage in no way broke 
down the barriers in this case. 

EXPERIMENTS IN 1913 

In the fall of 1912 the barberry bushes in the rust plat were sur- 
rounded with badly rusted wheat straw, the rust being in the teleuto- 
spore stage. In the spring of 1913 the aecidiospores were used in 
inoculating the four common cereals and einkorn. The results were 
surprising since it was supposed that the aecidia had been developed 
from the wheat-rust teleutospore sporidia. The results of the various 
trials are given below : 

Results of Inoculations of Cereals with Aecidiospores from Field Barberries 



Grain 


Trials 


Total 


1 


11 


III 


Wheat 


8 


o 

ii 5 


3 


1 3 
¥5" 


Oats 



417 









TOD" 


Barley 


3 2 


1 6 


1 8 
TTU 


6 6 


Rye 


40 
TT7 


7 
1 8 


^V 


5 5 
¥17 


Einkorn 


1 2 


2 

7 




1 4 



It will readily be seen that there would naturally be much doubt 
as to the origin of these aecidia from wheat-rust teleutospore sporidia. 
These aecidiospores were being used in spraying wheats in the rust 
nursery for the purpose of developing a rust epidemic. The results 
were very discouraging. There was a great deal of Agropyron repens, 
badly affected with the teleutospore stage of Puccinia graminis, near 
the barberry bushes. Experiments were therefore started to determine 
whether or not the aecidia on barberries might not have been devel- 
oped from this source rather than from the wheat-rust material. This 
view seemed all the more reasonable when it was observed that the 
Agropyron repens was very severely affected with the uredospore 
stage of rust, while the wheat, although it had been thoroughly and 
persistently sprayed with water containing the aecidiospores, had only 
a few scattered pustules of rust. 



BIOLOGIC FORMS 



25 



Barberry bushes which had been in the cellar all winter were set 
out in the field and covered with a heavy muslin cage. Badly rusted 
straw of wheat was tied around one bush and that of Agropyron repens 
around another. None of the check plants developed any aecidia while 
those surrounded with straw were very badly affected. The Agropyron 
repens material produced mature aecidia 10 days earlier than the wheat 
material and the aecidia were also developed in greater abundance. 

Results of Inoculations with Aecidiospores and Uredospores from Wheat 

AND Agropyron repens 



Grain 


A. repens 
aecidia 


A. repen? 
uredospores 


Wheat 
aecidia 


Wheat 
uredospores 


Wheat 


6 


3 
YT 


2 5 


3 


Oats 














Barley 


1 3 
7¥ 


2 7 
27 


2 4 
3"^ 


30 
3U 


Rye 


1 2 


3 7 

2 8 


7 


6 
"3U 



The field barberries were very probably infected with the rust from 
Agropyron repens, as will be readily observed by referring to the two 
tables. Further, the various biologic forms do not show any apparent 
change as a result of having been transferred to barberry, thus con- 
firming the results obtained in previous experiments. 

These inoculations, although not extensive, show quite clearly 
that Puccinia graminis tritici and Pnccinia graminis from Agropyron 
repens do not seem to develop any greater range of infection possi- 
bility for cereals after having lived for a time on the alternate host — 
the barberry. The incubation period, even on wheat, barley, and ein- 
korn, was a little longer in these experiments than that of uredo-devel- 
oped mycelium. 

Adaptation of Biologic Forms to New Hosts 
Magnus (1894, p. 362) was one of the first to suggest that a 
particular biologic form might, by constant association with one host, 
change its physiological capabilities to such an extent as to make a 
new race out of it. This view was also expressed by Dietel (1899, pp. 
81 and 113) who gave it as his opinion that a given rust formerly at- 
tacked a number of plants but by long association with one form be- 
came narrowed to this form more closely, possibly retaining also a 
somewhat weakened capability of attacking other forms. These 
authors distinguish between adaptation races (Gewohnheitsrassen) and 
true biologic forms, the tendency being, under favorable conditions, 



26 A STUDY IN CEREAL RUSTS 

for the former to develop into the latter. Eriksson (1902, p. 657) also 
expresses this view in a somewhat modified form. Ward (1902-1) 
shows that adaptation of Puccinia dispersa takes place. Klebahn (1904, 
pp. 152-167) cites numerous experiments to show that this may be 
the case. Miss Gibson (1904, pp. 184-191) grew a number of suc- 
cessive generations of rust on resistant host varieties, but, in that time, 
found little adaptational tendency. Massee (1904, p. 17) explains the 
resistance and susceptibility of various plants to parasitic fungi on the 
ground of the presence or absence of chemotactic substances in the 
host. He contends that saprophytes can be educated to become para- 
sites. This would also apply in large measure to biologic forms. Sal- 
mon (1905, p. 183) grew Erysiphe graminis from wheat on Hordeum 
sylvaticum for five generations and found no diminution in the power 
to infect the original host. Freeman and Johnson (1911, p. 28) con- 
clude that "the host plants exercise a strong influence, not only on 
the physiological and biological relationships, but in some cases even 
on the morphology of the host." 

An attempt was made to test this matter. The object was to de- 
termine the change, if any, in the physiology of the rust as evidenced 
by its power of infection and in its morphology as evidenced by changes 
in spore dimensions. For this purpose Puccinia graminis tritici was 
grown on Minnesota No. 163, a susceptible wheat, and on einkorn 2433. 
The einkorn, in the first few trials, was apparently one of the most 
resistant of the Triticums to the wheat stem rust. The rust was trans- 
ferred to einkorn and grown on this host through successive genera- 
tions for 19 months, transfers being made, on an average, once every 3 
weeks. After one year on einkorn the rust seemed to be much more 
virulent than the original wheat rust had been. Einkorn plants were 
inoculated with this einkorn rust and with uredospores from wheat. 
In both cases there was 100 per cent of infection, but the virulence of 
the infection was quite different. On the leaves inoculated with rust 
from einkorn, leaf areas one to two centimeters long were affected, 
being well covered with pustules shedding spores in great abundance. 
Individual pustules were from one to three millimeters long and nearly 
as broad, giving every indication of a severe rust attack. On the plants 
inoculated with wheat rust, on the other hand, the pustules were al- 
ways smaller, although fairly numerous. Few of them were as much 
as one millimeter long and many of them did not rupture at all. 

Some wheat plants were inoculated with rust from einkorn and 
others with the rust taken directly from wheat. Here again there was a 
considerable difference in virulence of infection. The pustules developed 
from einkorn-rust inoculations were fairly large and numerous. Some 
of them were two millimeters long, although the average length was 
less than this. The infection on wheat inoculated with wheat rust was 



BIOLOGIC FORMS 27 

much more severe. Areas of the leaf two centimeters in length were 
often almost covered with pustules, some of which attained a length of 
seven millimeters. 

A number of other trials were made, the last after the rust had 
been confined to einkorn for 17 months. In one experiment the re- 
sults were just the opposite of what were expected. This is explained 
by the fact that lack of greenhouse space necessitated keeping the ein- 
korn plants inoculated with einkorn rust in a draught of cold air. 
There were only a few wheat uredospores available in inoculating the 
wheat, so, in this experiment, einkorn-by-einkorn inoculations were not 
so successful as einkorn-by-wheat. In subsequent trials, however, un- 
der uniform conditions, the results first described were substantiated. 

The conclusion, then, is justified that by confining Puccinia gram- 
inis tritici to einkorn for successive generations throughout a year or 
more the rust adapts itself somewhat to its new host and loses, at least 
to a slight degree, its power to infect the original host. It would no 
doubt require a very long period of time to fix this character in the 
plant to such a degree as to make it a new biologic form. But there is 
very evidently such an adaptational tendency (see Plates II and III). 
It must be noted, however, that this new character is not so firmly 
fixed that it cannot be overbalanced by environmental factors. The ex- 
perimental production of new forms is apparently possible, but a long 
period of time is required. 

The change in the fungus manifests itself not merely in the para- 
sitic tendency toward the host but in the morphology as well. Wheat 
and einkorn were inoculated with spores from the same plant. The 
uredospores after growing for a year on wheat averaged 37.85x22.76/^ 
while those grown on einkorn for a year measured 33.58x21.79 M. 
When einkorn was inoculated with Puccinia graminis tritici aecidio- 
spores, the resulting uredospores were more nearly identical with the 
wheat-rust spores in length. The width, however, remained practically 
the same. The average size of these spores was 35.92 x 21.69 yu, 

SUMMARY OF PART I 

1. Direct transfers of Puccinia graminis have been made from 
oats to both wheat and rye. The rusts from oats and barley used in 
these experiments could be transferred to rye more easily than those 
used by Carleton or those used by Freeman and Johnson. The barley 
rust, however, did not prove as versatile as the strain used by Freeman 
and Johnson. 

2. The use of anesthetics has some effect in rendering an immune 
plant slightly more susceptible to the rust; leaf injury apparently had 
no effect. 

3. High fertilization, by increasing the virulence of the attack 



28 A STUDY IN CEREAL RUSTS 

on semi-immune forms, may have some influence in breaking down 
biologic forms. 

4. There appears to be a physiological and even a slight mor- 
phological change in the rust fungus when grown continuously on a 
semi-immune host. The physiological change manifests itself as an 
adaptation to the new host, which, however, is very gradual. 

5. There are indications that biologic forms of cereal rusts, at 
least Puccinia graminis tritici, do not lose their specialization tenden- 
cies when grown on barberry. 

6. The degree of incompatibility of host and parasite varies 
greatly. In semi-compatible forms, fairly large leaf areas are some- 
times killed, indicating a killing of host cells by fungus and consequent 
death of the mycelium itself. In this respect they resemble very closely 
some of the rust resistant forms of wheat. The biologic forms of rusts, 
therefore, with susceptible and immune varieties of host plants, throw 
light on the question of the nature of resistance to Puccinia graminis. 



PART 11. RUST-RESISTANT VARIETIES OF WHEAT 

HISTORICAL 

It has long been a matter of common observation that some wheats 
are more resistant than others to the attacks of Puccinia graminis and 
other rusts. Among the earher observers Henslow (1841, p. 3), La 
Cour (1863, p. 326) and Little (1883, p. 634) note that some wheats 
are less injured by rust than are others. Bolley (1889, p. 16) observes 
that those varieties least susceptible to rust are "hardy, stiff-strawed 
wheats, having smooth, fibrous leaves." Anderson (1890, p. 84) says 
that hard, flinty wheats are more rust-resistant than others. He thinks 
it may be due to the large amount of silica in the hard wheats. Cobb 
(1892) advanced his "mechanical theory" to explain resistance. It 
was due, according to his idea, to morphological characters of the 
host, namely, thick cuticle, waxy coating, and small stomata. Hitch- 
cock and Carleton (1893, p. 12) also correlated resistance with morpho- 
logical characters, asserting that resistant forms had stiff, upright 
leaves with a thick epidermis. Eriksson (1895, p. 199) and many 
others since have shown, however, that a wheat resistant to one spe- 
cies of rust is not necessarily resistant to another species, thus indicat- 
ing a rather delicate relationship as the basis of resistance. 

Eriksson and Henning (1896, pp. 332-365) were unable to sub- 
stantiate Cobb's mechanical theory, since morphological characters 



RUST-RESISTANT VARIETIES OF WHEAT 29 

could not always be correlated with resistance. They suggest that re- 
sistance is of a complex chemico-physiological nature and is inherent 
and fairly constant within the plant. Ward (1902) decid"ed in connec- 
tion with Puccinia dispersa on the bromes that resistance was indepen- 
dent of any recognizable morphological character and suggested that 
the problem was much the same as that dealing with the factors gov- 
erning fertility and sterility of stigmas to pollen. Biffen (1907, p. 128) 
concludes that resistance to Puccinia glmnarum is independent of any 
discernible morphological character. He reasserts this principle in a 
later work (1912, pp. 421-429). Bolley (1908). although not positive, 
inclines to the view that disease-resistance is physiological rather than 
morphological in its nature. Cook and Taubenhaus (1912) show that 
various vegetable acids are toxic to parasitic fungi and that the amount 
of some of these acids present depends on the stage of ripening of the 
fruit. Jones, Giddings, and Lutman (1912, p. 83) conclude that the 
resistance of potato tubers and leaves to Phytophthora infesfans is due 
to something either largely or wholly within the tissues. The consen- 
sus of opinion among the more recent investigators seems to be that 
there is a very delicate balance maintained between host and parasite. 
This balance is dependent, to a certain extent, on the environmental 
conditions under which host plants are grown. This, however, as well 
as other phases of the problem, will be discussed more fully under the 
various sections into which the question naturally subdivides itself. 

FORMS WHICH ARE RESISTANT 

Attention has frequently been called by various observers to the 
fact that freedom from disease does not necessarily indicate resistance. 
Varieties which mature early may escape the disease, and various other 
factors, mainly ecological, may influence the degree of resistance. 
Careful experiments, however, have shown that some varieties of 
Triticum durum are really resistant. The resistance of durum wheats 
varies with locaHty according to RoUey's observations (1906, p. 662). 
Consequently to determine absolute resistance the plants should be 
grown under controlled conditions. Further, they should be subjected 
to conditions favorable for infection. 

Field Observations 
Field observations were made on various forms which were grown 
in a rust plat. An epidemic was induced by spraying frequently with 
water containing a large number of spores of Puccinia graminis tritici. 
Frequent observations were made on the amount of rust. Final notes 
were taken at ripening time. The percentage indicates in each case 
the estimated percentage of resistance, assuming absolutely immune 
forms to have a resistance of 100 per cent. 



30 A STUDY IN CEREAL RUSTS 

Estimated Percentages of Rust on Varieties Grown in 1911 and 1912 



1911 



1912 





Per 






Per 


Variety 


Cent 


Variety 




Cent 


Einkorn 2433 










, Dickinson 1910 Fed. 


No. 4. . 92 


Minnesota No. 


169 


15 


No. 6 


90 


lumillo 1736 .. 




98 


No. 7 


93 


Minnesota No. 


188 


30 


No. 8 


.95 


Einkorn 2433 . 




96 


lumillo 1736 


95 


Khapli 




95 


Kubanka 1516 No. 8. . 


15 


Minnesota No. 


169 


35 


Kubanka 1516 No. 9. . 


10 


Arnautka 288 




97 


Minnesota No. 163 


10 


Arnautka 1431 




67 



It will be noticed that in 1912 the resistance was slightly greater 
than in 1911. This may be accounted for by the fact that in 1911 the 
grains were sown late, giving the rust ample opportunity to develop 
fully. 



EXPERIMENTAL 



Greenhouse Trials 



The varieties tested for resistance in the greenhouse were : Minne- 
sota No. 163, Minnesota No. 169, Kubanka 1516, Nos. 8 and 9, Ku- 
banka 2094, lumillo 1736, einkorn 2433, Nos. 4, 6, 7, and 8, emmer 
1522, Arnautka 288, and Khapli. Of these, Minnesota No. 163, Min- 
nesota No. 169, Kubanka 1516 did not prove resistant. The behavior 
of einkorn has already been discussed under adaptation of biologic 
forms. Very careful observations were made on the others to deter- 
mine as accurately as possible their comparative resistance. The inocu- 
lations were made with fresh, viable spores and the plants put under 
bell jars 48 hours after inoculation. 

The incubation period varies with temperature conditions, both 
high and low temperatures lengthening the period very perceptibly. 
No experiments with this particular object in view were made, but 
numerous observations brought the fact out very clearly. Under the 
same conditions on Minnesota No. 163 the incubation period is shorter 
than on any of the resistant forms. With an average temperature of 
about 65 degrees Fahrenheit and a variation of from 40 to 75 degrees 
and a relative humidity of about 55 per cent, pustules appear on Minne- 
sota No. 163 in 7 or 8 days. In case of lumillo the period is usually 
about 2 days longer, although considerable variation was found. Em- 
mer has an incubation period of about 11 days, Arnautka, 12 days, and 
Khapli, 14 days. Arranged in order of their susceptibility these vari- 
eties are as follows : 



RUST-RESISTANT VARIETIES OE WHEAT 31 



Variety Incubation Period 

Minnesota No. 163 7 days 

lumillo 1736 9 days 

Emmer 1522 11 days 

Arnautka 288 12 days 

Khapli 14 days 

It will thus be seen that the incubation period is longer on the more 
resistant varieties. These figures are, of course, not absolute, but vary 
with the temperature, and, to some extent, with soil conditions. All 
however, vary in nearly, but not absolutely, the same proportion. 

The character of infection is distinctly different on the different 
varieties. It is quite noticeable that the same phenomena are observed 
as appear on various biologic forms. On Minnesota No. 163 the pus- 
tules are large, varying from 2 to 6 millimeters in length. They rup- 
ture the epiderm very readily and shed spores in great abundance. 
Very rarely are small, unruptured pustules developed. The host tissues 
nearly always remain fairly healthy, a yellowing which gradually ap- 
pears furnishing the external evidence that the fungous hyphae are in 
the plant. On einkorn and lumillo, which in the greenhouse are only 
fairly resistant, the pustules are usually somewhat smaller. There is 
a tendency in these two forms toward the development of small dead 
areas. These areas are either very distinctly yellowed or sometimes 
killed outright. The general appearance is, however, usually not 
sharply different from that of infected Minnesota No. 163, except in 
degree of infection as evidenced by smaller pustules on einkorn and 
lumillo. On emmer there were often long infected, yellow areas in 
which there was a fairly large number of very small pustules, usually 
less than one millimeter in length, many of which never ruptured. 
Then again fairly large areas of host tissue were practically killed and 
only a few small green islands developed. On both Arnautka and 
Khapli, areas from one to two centimeters long were killed, the leaf 
appearing white and dead (see Plate VI, A). In these areas there was 
often a moderately large number of "green islands" with very small, 
unruptured pustules in the centers. When the pustules did rupture they 
were always very small, seldom, if ever, exceeding one millimeter in 
length and often being mere dots. The large areas involved can be 
explained rather on the basis of multiple infection than on the basis of 
the spreading of the mycelium from a few infections. Histological 
examination of diseased areas verifies this supposition. 

The spores on the resistant varieties were smaller than on Minne- 
sota No. 163. Spores of Minnesota No. 163 averaged 35.38x21.39 /<. 
while those of emmer were 33.04x21.30 pi. The Khapli spores were 
smallest, being only 29.69 x 20.68 pi. It was found that spores from 
different pustules varied somewhat in average size. Therefore spores 



iZ A STUDY IN CEREAL RUSTS 

from a number of pustules were measured in determining the averages. 
The fact that pustules are produced only with difficulty and that the 
spores are smaller on resistant varieties would seem to indicate that 
the fungus is not vigorous and cannot develop extensively although it 
may gain entrance into the leaf tissues. 

COMPARATIVE VIRULENCE OF AECIDIAL AND LONG-TIME 
UREDOSPORE INOCULATIONS ON RESISTANT FORMS 

Various observers have remarked on the reinvigorating power of the 
aecidial stage of Puccinia graminis. Plowright (1882, p. 234) was of 
the opinion that much more damage was done by aecidial infections 
than by infection by uredospores which had been reproduced for a 
number of successive generations. Bolley (1889, p. 13) states that the 
aecidium, being a sexual product, should be considered as functionally 
reinvigorating. He also reasserts this principle in a later work (1909, 
p. 182). Arthur (1902. pp. 68 and 69 and 1903, p. 17) observes that 
primary uredospores have a greater disturbing effect on the host than 
do long-time uredospores. Freeman and Johnson, on the other hand, 
cite experiments (1911, p. 33) to show that when the aecidial and 
teleutospore stages were excluded for 52 generations the fungus still 
retained its power of infection. The fact that sexuality in the rusts has 
been definitely established would make it seem reasonable to suppose 
that there would be a reinvigorating power. However, Barclay ( 1892, 
pp. 8 and 40) states that in India there are no barberries for "enormous 
distances" from fields of wheat in which Puccinia graminis is quite 
destructive. McAlpine (1906, p. 58) points out that Puccinia graminis 
probably causes no more damage in any country in the world than it 
does in Australia where barberries are practically absent and aecidia 
have never been found. The rust is quite serious in South Africa, but, 
according to Pole Evans (1911), the aecidial stage is absent. 

Comparative trials were made with aecidiospores, primary ured- 
ospores, and long-time uredospores. The varieties used were not in 
all cases the most resistant, since no seed of some of the more resist- 
ant forms was available when the aecidia appeared. Trials were made 
on Minnesota No. 163, Kubanka 1516, lumillo 1736. and einkorn 2433. 
The two last-mentioned forms are fairly resistant. The long-time ured- 
ospores used represented the twenty-fourth generation on wheat. A 
number of trials were made and the results were not alwavs uniform. 
The incubation period of the fungus when developed from primary 
uredospore or from aecidiospore inoculations was slightly longer than 
when developed from long-time uredospores. The pustules developed 
from long-time uredospores were apparently smaller and more numer- 
ous, while those from aecidiospores and primary uredospores averaged 



RUST-RESISTANT VARIETIES OF WHEAT 33 

somewhat larger, were deeper brown, and seemed to be shedding 
spores in greater abundance. On einkorn, in one experiment, the re- 
sults were directly opposed to this. The differences were not especially 
striking, the aecidial infections being perhaps slightly more virulent. 
There is considerable evidence that the virulence of the rust attack 
when carried by aecidiospores or primary uredospores is exceptionally 
virulent. The results of these experiments, however, would not justify 
such a conclusion in this particular case. 

METABOLISM OF THE HOST AND RUST RESISTANCE 

There seems to be no question but that weather and soil condi- 
tions, determining the metabolism of the host plants, exert an influence 
un the prevalence of rust in the field. Little (1883, p. 634) states Uiat 
weather is the determining factor and adds that high manuring, espe- 
cially with nitrogenous manures, predisposes wheat plants to rust. 
Bolley (1889), Anderson (1890), and many others since have held that 
this is the case. Bolley suggests as a possible cause the increased 
evaporation and consequent raising of ^the relative humidity. Jones 
(1905) shows that Phytophthora rot of potatoes tends to be more seri- 
ous after a heavy application of nitrogenous fertilizers to the land. 
Miss Gibson (1904) concluded as the result of experiments with the 
chrysanthemum rust that in an almost immune form normal develop- 
ment of rust does not depend on the state of health of the plant, but 
that a luxuriant state of growth favors the development of the fungus. 
Hennings (1903, pp. 41-45), on the other hand, states that in observ- 
ing plants infected with perennial smuts and with rusts he found that 
the disease disappeared when the plants were placed under the most 
favorable cultural conditions. This is not in accord with Arthur's gen- 
eralization (1903, p. 13) that "so intimate is the association of host 
and parasite that as a rule the vigor of the parasite is directly propor- 
tional to the vigor of the host." /Vpparent discrepancies may, how- 
ever, be explained by the fact that different plants and different para- 
sites react quite differently. As far as the rusts of cereals are con- 
cerned, Arthur's generalization would seem to be correct. Biffen 
(^1912, pp. 421-429) shows that Puccinia glumanim is most virulent 
when a complete fertilizer is used and that the virulence decreases 
with the decrease in amount of fertilizer. 

Less work has been done to determine the exact manner in which 
these causes operate. De Bary (1887, p. 359) says, "The physiological 
reason for these predispositions cannot in most cases be exactly stated ; 
but it may be said in general terms to lie in the material composition of 
the host, and therefore to be indirectly dependent on the nature of its 
food." Marshall Ward (1902, p. 145), in experiments with Puccinia 
dispersa on bromes, tried the effect of mineral starvation and concluded 



34 A STUDY IN CEREAL RUSTS 

that "lack of minerals in no way secured immunity from infection, 
although seedlings deficient in phosphorus or nitrogen tended to show 
retardation of infection." The well-nourished plants produced more 
spores than the underfed ones. This seems to be due not so much to 
the presence or absence of any particular chemicals and a direct effect 
on the fungus but rather to the effect on the host. However, attempts 
have been made to prevent diseases, among them rusts of cereals, by 
adding various substances to the soil. Anderson (1890, p. 84) recom- 
mends the use of salt, iron sulfate, a;nd lime as tending to decrease the 
amount of rust. Galloway (1893, p. 208) tried the effect of flowers of 
sulfur, potassium sulfid, ammonium carbonate, potassium bichromate, 
calcium hydroxid, and iron sulfate when applied to the soil, but found 
them of no particular value in preventing rust. Laurent (1902, pp. 
1040-1042) concludes that potatoes can be immunized against Phytoph- 
ihora infestans by treating the soil with copper sulfate. Marchal (1902, 
pp. 1067 and 1068) tried the effect of copper sulfate and iron sul- 
fate of various strengths when added to Sach's solution, on the se- 
verity of attack of Bremia lactucae on lettuce. He found that by add- 
ing 4 or 5 parts of copper sulfate to 10,000 parts of Sach's solution 
he was able to render the plants practically resistant to the fungus, 
}et leave the vegetation normal. In experiments attempting to immun- 
ize cereals to rusts he was unsuccessful. Massee (1903, p. 142) pre- 
vented the development of fungi on tomatoes and cucumbers by water- 
ing them with copper sulfate solution. This did not give the desired 
results with Oidium on barley. He states that not all plants can be 
treated in this way without endangering their health. Chemical analy- 
sis of treated and untreated tomatoes showed that there was no more 
copper sulfate in treated and therefore immune plants than there was 
in those which had received no treatment. Massee suggests therefore 
that the copper sulfate reacts with certain substances in the soil, thus 
indirectly conferring immunity. Freeman and Johnson (1911, pp. 69- 
70) call attention to the complexity of the problem and the need for 
differentiating results. 

EFFECT OF WATER CONTENT OF SOIL 

Statements to the effect that low-lying, wet soils predispose cereals 
to rust are frequently made in the literature of the subject. Reference 
has already been made to some of these. It was observed very fre- 
quently in experiments mentioned in this paper that when the relative 
humidity was high infection was not only surer to result but that it 
was also more severe. It was therefore thought worth while to de- 
termine whether a high water content of the soil would act as a pre- 
disposing factor. 

The varieties used were: Einkorn 2433, Kubanka 1516, lumillo 



RUST-RESISTANT VARIETIES OF WHEAT 35 

1736, and Minnesota No. 163. There was no difference in the amount 
of water until the plants germinated. Immediately after germination, 
however, the soil in one series was kept very wet while that in another 
series was kept as dry as was possible without endangering the life of 
the plants. The soil in the wet series had a water content of 31.35 
per cent, while that of the dry series was 6.16 per cent at the conclusion 
of the experiment. Repeated trials were made with substantially the 
same results. 

The number of einkorn and lumillo leaves which became infected 
in the wet series was smaller than in the dry series. The percentages 
for the others were practically the same. In virulence of infection, 
however, there was considerable difference. The varieties also reacted 
somewhat differently so each will be considered separately. 

On Kubanka the virulence of infection, especially in the early 
stages, is very markedly inferior on the plants in the wet series. The 
pustules during the early stages are often small and on some plants 
do not appear at all, the leaf merely becoming yellow. Later the plants 
in dry soil were often completely covered with large, vigorous pus- 
tules while those in wet soil, although producing a moderately large 
number of pustules, were not nearly so badly affected. In both series 
there were many secondary infections along the leaf. There was a 
distinct tendency in the wet series toward leaf-yellowing. It was at 
first thought that the mycelium might be spreading through the tissues. 
Histological examination, however, failed to confirm this supposition. 
Apparently it was only a slight chlorotic condition due to excessive 
water content. The infection was unquestionably more severe on plants 
grown in dry soil (see Plate IV). 

On einkorn the differences were not so sharp, although there ap- 
peared to be a slightly more severe infection on plants in the dry series 
than on those in the wet series. The rust appeared at about the same 
time, the virulence of infection being at first quite distinctly greater 
on the dry-soil plants. Later this difference was not quite so marked, 
although still apparent. 

The sharpest difference was on lumillo. Only a few leaves in the 
wet series were really badly infected, while those of the dry series 
showed a surprisingly virulent attack. There is no question but that 
the infections secured on plants in these dry series were more severe 
than were those on any other lumillo plants inoculated during the 
various trials with this variety. It is not often that a really vigorous 
development of the fungus occurs on lumillo, but when the water con- 
tent of the soil is very low, the infection at times shows surprising vir- 
ulence (see Plate V). 

The results on Minnesota No. 163 varied more than those of the 
other forms. The pustules in nearly every case were large and vigor- 



36 A STUDY IN CEREAL RUSTS 

ous with but little difference between the two series. There seemed to 
be a tendency for the mycelium to spread more in the plants grown in 
wet soil, but the pustules were not larger than those on dry-soil plants. 
Whatever difference there was appeared as a slightly greater virulence 
on the wet-soil plants. 

It will Ije noticed that lumillo and Kubanka, drought-resisting 
plants, were more severely attacked when grown in dry soil. Minne- 
sota No. 163, on the other hand, a mesophyte, did not show so much 
difference. It is probable then, that, conditions having been favorable 
for a rust infection, the water relation in the soil which is most favor- 
able for the host plant's development is also the most favorable for the 
development of the rust. It seems probable also that in at least some 
forms it is not the water content of the soil which predisposes grains 
growing in low places to rust but rather the increased relative humidity 
which enables the rust spores to germinate and infect the plants. The 
temperature in such places also probably exerts an influence. This is 
pointed out by F"reeman and Johnson (1911, p. 65) in connection with 
the rust epidemic of 1904. 

EFFECT OF FERTILIZERS 

In the first series the varieties used were : Minnesota Xo. 163, 
Arnautka 288, Khapli, emmer 1522, lumillo 1736, einkorn 2433, and 
Kubanka 2094. Some were planted in ordinary rich loam, others in 
rich loam plus fresh barnyard manure, and a third series in rich loam 
to which barnyard manure and bone meal had been added. Especial 
care was taken to keep all plants under the same conditions of tempera- 
ture, moisture, and light both before and after inoculation. 

On the wheat plants which were grown on very heavily fertilized 
soil the infection was clearly more severe. The infected areas were 
very large as were also the individual pustules. The most severely 
attacked plants were in one pot which had been fertilized with manure 
and bone meal. Aside from this one pot, however, there was but little 
variation among the fertilized pots. The infection on the check plants 
remained inferior, although it was very vigorous. 

Similar results were obtained from einkorn and lumillo plants. In 
the' case of these two forms the plants in the manure-and-bone series 
developed the worst rust attack, the manure was next, and the plants 
in ordinary soil were more lightly attacked. It should be remarked that 
the differences were not strikingly sharp, although they were quite 
apparent. Emmer and Arnautka gave no distinct results. There was 
a great deal of variation in the individual pots and no one series stood 
out clearly from the other two. 

The Kubanka and Khapli plants showed some differences. The 
character of infection is very different from that of the other forms. 



RUST-RESISTANT VARIETIES OF WHEAT 



37 



In the case of the former the plants grown in pots to which barnyard 
manure had been added were most severely attacked, while there was 
little difference between those grown in loam and those manured with 
both barnyard manure and bone meal. Khapli plants grown in soil 
fertilized with both manure and bone were fairly successfully infected. 
Areas of the leaf, one centimeter in length, were sometimes infected, 
and pustules as big as a pin head were developed. There was little dif- 
ference between the other pots, the infection being somewhat milder 
than in the heavily fertilized ones 

It is quite probable that there was available in the rich loam very 
nearly all of the plant food the plants were capable of utilizing. This 
would account for the fact that the differences were not always greater. 
On the whole it might be concluded that very heavy fertilization is 
somewhat conducive to increased severity of attack on very resistant 
varieties as well as on susceptible forms. 

Since the check plants in the trials just described were grown un- 
der such favorable conditions, it was determined to grow the checks in 
poorer soil in the next series. Therefore they were planted in mod- 
erately fine sand (S) (see table below) to which but a very slight 
amount of leaf-mold had been added. Nitrogen (N) was added to 
another series in the form of calcium nitrate, to a third was added cal- 
cium phosphate (P), and the fourth received both calcium phosphate 
and calcium nitrate (P and N). The salts were applied in water. The 
plants were watered three times with distilled water containing the 
proper salt or salts, at the rate of 3 grams per 500 cc. The same varie- 
ties were used as were used in the preceding series. 

Observations on results gave the following order of virulence, the 
first being most virulent and the others arranged on the same basis. 
Two observers took notes with the following results : 

Effect of Fertilizers on Virulence of Rust Attack 





Order of Virulence 


Grain 


1 


2 


3 


4 


Einkorn 


N 


PandN 


P 


S 


Emmer 


PandN 


N 


P 


S 


Kubanka 


PandN 


N 


P 


s 


Khapli 


PandN 


N 


P 


s 


Arnautka 


N 


S 


P 


PandN 


lumillo 


N 


P 


S 


P and N 



The somewhat conflicting results suggested the desirabilitv of an- 
other trial. Four series were arranged as follows: Pure sand (S), 
ordinary field soil (O), sand plus nitrogen (N), and sand plus phos- 
phorus (P). The nitrogen and phosphorus were added as in the pre- 
ceding experiment. Three persons working independently made ob- 



38 



A STUDY IN CEREAL RUSTS 



servations, but there was no great difference of opinion in any case. 
The order in which different observers placed them was sometimes dif- 
ferent, showing- that there was sometimes Httle choice among the 
various pots. The results are given in the following table : 

Effect of Fertilizers on Virulence of Rust Attack 



Grain 


Order of Virulence 


Remarks 


1 


2 


3 


4 


Emmer 

Kubanka 

lumillo 

Khapli 

Arnautka 

Wheat 


N 
S 
N 
S 
P 

s 


P 
P 


N 
S 

N 


S 
N 
P 
P 
N 
P 





s 








Einkorn 

Einkorn 

Emmer 

Kubanka 

lumillo 

Khapli 

Arnautka 

Wheat 


N 

P 

N 
S 
N 
S 
P 
N 


P 

N 
S 
P 
P 

N 
N 
S 


S 

S 
P 

N 

P 

s 
P 








s 






Little difference between N and P 

Little difference between P and S 

Distinct 

Little difference between P and N 

Little difference between P and N 

Little difference between N and S 

Little difference between S and P 



In this experiment the two most resistant forms available for study 
were watched very carefully. Both Kubanka 2094 and Khapli proved 
to be very resistant even when very highly fertilized. It is a rather 
striking fact that in both cases plants grown in sand showed a slightly 
more virulent infection. The differences were not great. In fact, it 
was often hard to decide which plants were most severely affected. It 
was observed that plants which had been under the most favorable con- 
ditions for infection were most severely attacked regardless of the fer- 
tilizer used. The difference in conditions was due to the fact that there 
were not enough bell jars to cover all the plants, so some were placed 
in tubs containing water on the bottom. They were then covered with 
pieces of glass. The films of moisture were not so persistent here as 
under the jars, and the difference in the amount of rust was quite 
marked. This was taken into account in determining results and mak- 
ing comparisons. 

If these plants had been allowed to grow longer it is quite prob- 
able that those fertilized with nitrogen would have become more se- 
verely rusted while those fertilized with phosphorus would have been 
slightly less severely affected. This would seem to be due not to the 
specific action of the chemicals on the rust fungus but rather to their 
effect on the general condition of the plant, and, in the field, on the 



RUST-RESISTANT VARIETIES OF WHEAT 39 

immediate atmospheric conditions. The direct effect of chemicals in 
the soil on the amount of rust on resistant varieties is not great, only a 
slight quantitative difference being apparent. 

In order to determine by more accurate methods whether or not 
there was a direct effect of substances in the soil on the amount of 
rust (1) fungicides were put into nutrient media, and (2) certain 
nutrient salts were used in amounts varying from deficiency to excess. 

Sach's modified culture solution was used and to this one per cent 
of agar was added. The series was arranged as follows, the amounts 
of nitrate and phosphate being the variables : 

I. Potassium nitrate; 2 grams per 1,000 cc. 
II. Calcium phosphate ; 3 grams per 1 ,000 cc. 

III. Potassium nitrate; .05 grams per 1,000 cc. 

IV. Calcium phosphate; .075 grams per 1,000 cc. 
Minnesota No. 163 wheat was used. After inoculation plants were 

all placed under bell jars and kept under uniform conditions. Arranged 
according to virulence, the most severely affected being placed first, 
they would be arranged as follows: III, II, IV. I. 

This was tried a second time with exactly the same results. The 
plants appeared about the same, all growing fairly well in the agar. 
All were well infected, producing a fairly large number of large, 
healthy pustules. They were kept three weeks after inoculation and 
by this time there was not much difference between II, IV, and I, but 
III was still much more virulently attacked. It seems, therefore, that 
an excess of nitrogen does not necessarily, in itself, cause an increase in 
the amount of rust and an excess of phosphorus does not affect it very 
appreciably. 

The effect of excluding nitrogen and phosphorus was next tried. 
Sach's modified medium plus one per cent of agar was again used and 
in I no potassium nitrate was added while calcium phosphate was ex- 
cluded from II. The plants in I were lighter colored from the first 
than either those in II or the checks. They were inoculated six days 
after planting. A good, vigorous infection resulted, the plants in I 
being slightly more severely attacked than those in II. The leaves of 
I began to turn yellow after three weeks, and the rust did not spread 
farther. The check plants were more severely attacked than those in 
either I or II. Here again, however, the differences were not very 
great. There was a slight quantitative difference but qualitatively there 
v/as scarcely any difference. This is in keeping with Ward's conclu- 
sions reached after his work on mineral starvation, reference to which 
has already been made. 

An attempt was then made to determine whether it was possible 
to confer immunity by means of various salts. Copper sulfate, copper 
carbonate and iron sulfate were used in strengths varying from 1 to 



40 A STUDY IN CEREAL RUSTS 

10,000 to 1 to 2,000. They were added to Sach's medium in these pro- 
portions. Minnesota No. 163 was used for all the trials. Copper sul- 
fate could not be added in larger amounts than 1 to 5,000, since it 
stunted the plants when more was added. Copper carbonate could not 
well be used in greater concentration than 1 to 2,000. Iron sulfate 
did not dwarf the plants when used at the rate of 1 to 2,000. None of 
the solutions used diminished the amount of rust very appreciably when 
used in such concentration as to permit of normal development of the 
host plant. There was a. slightly smaller amount of rust on plants 
grown in the medium to which copper sulfate had been added in 
amounts of 1 to 4,000 and 1 to 2,000. However, a very good infection 
v\^as secured on all of them, even on those which never grew to a greater 
height than one inch. Neither was there any less mildew on any of 
the plants. None of these substances, apparently, can immunize wheat 
against rust, even when used in such concentration as to dwarf the 
plants to one-sixth their normal size. 

These experiments show that in the case of Puccinia graminis, 
since it is a very highly specialized, obligate parasite, there is a very 
intimate relationship between host and parasite, and whatever is con- 
ducive to the health of host is ordinarily conducive to the vigorous de- 
velopment of the parasite also. This applies not only to susceptible 
forms but also to forms uncongenial to a biologic form and to resistant 
varieties. 

THE NATURE OF RESISTANCE 

The work of Cobb, Eriksson, Ward, and others on the nature of 
resistance has already been mentioned. The theory which Ward de- 
duced from his extensive work on parasitism was that there are en- 
zymes or toxins and antitoxins produced by host or parasite or both. 
His work on "A Lily Disease" (1888) showed that in all probability 
Botrytis secretes an enzyme which enables it to live more successfully 
on the host. Pfeffer had already given the name chemotaxis to the at- 
traction certain substances seemed to have for certain growing plant 
parts. Miyoshi (1894, p. 21) claimed to have been able to observe a 
very definite chemotropism when a Tradescantia leaf was injected 
with a wheat-leaf decoction and then inoculated with Uredo linearis 
{Puccinia graminis). The same author decided (1895) that a large 
number of fungi responded to chemical attraction. Massee (1904, 
p. 7) attached a great deal of significance to chemotaxis. He asserted 
that infection depended on the presence in the plant cell of positive 
chemotactic substances and further that "in the future we shall be justi- 
fied in defining an immune plant as an individual in which the positive 
chemotactic substance, necessary for facilitating the entrance of the 
germ-tubes of a given parasitic fungus into its tissues, is absent." On 



RUST-RESISTANT VARIETIES OF WHEAT 41 

the other hand Errera (1892, p. 373) contends that so-called chemotro- 
pism is in many cases merely positive or negative hydrotropism. Ful- 
ton (1906, pp. 81-107) says that there is no definite chemotropic re- 
sponse on the part of fungi. Nutrient solutions cause marked growth, 
in his opinion, but cause no definite turning of hyphae in their direc- 
tion. Hydrotropism, however, was observable. 

The behavior of the germ tube of Pucciuia graminis and its en- 
trance into the host plant has been described and figured bv various 
authors. Ward (1881-1, p. 217) figures it as forming a slight swell- 
ing and then growing directly into the tissues of the host. Bolley 
( 1889, p. 14) shows the germ tube growing directly through the stom- 
atal opening and branching out between the mesophyll cells. This has 
never been seen by the writer. An appressprium always formed in all 
cases of infection observed! Pole Evans (1907, p. 445) describes and 
figures normal infection quite completely. 

A considerable amount of work has also been done on determining 
the fate of germ tubes when they are produced on immune host plants. 
Klebahn noted (1896, p. 263) that sporidia of Pnccinia convallariae- 
digraphidis could bore through the epidermal walls of Polygonatmn 
midtiflorum, an uncongenial host, but that the germ tubes developed no 
further. He concludes that infection is of the nature of a conflict be- 
tween host and parasite. Ward (1901 and 1902) showed in connection 
with Pnccinia dispersa on bromes that the germ tube might enter and 
cause normal infection, the mycelium might develop and never produce 
pustules, or the tissues of the host plant might be killed very early, 
thus precluding the possibility of much development on the part of 
the fungus. The same author further shows (1904, p. 29) that in 
normal infection of a susceptible species of Bromus the host cells 
retain life for a surprisingly long time. Miss Gibson (1904) examined 
the leaves of a large number of plants, widely separated taxonomically, 
which had been inoculated wnth spores of Uredo chrysanthemi. She 
found that the germ tubes might enter the plant tissues very readily 
but never formed any hausloria and consequently no pustules. 
Furthermore the hyphae usually <Iied when t'^ey came in contact with 
a cell. On resistant varieties of Chrysanthemum it was found that 
haustoria might develop, but areas of host tissue in the neighborhood 
of the hyphae were killed, thus preventing the further spread of the 
fungous mycelium. Her conclusion is to the efifect that when the 
germ tube of a uredine fungus enters any but its proper host plant a 
struggle goes on, resulting in the death of the host locally and of the 
parasite. The closer the relationship between the plant and the proper 
host of the rust the longer and more extensive will be the struggle. 
Salmon (1905) found that when barley was moculated with spores 
of Erysiphe graminis from wheat incipient haustoria might be formed 



42 A STUDY IN CEREAL RUSTS , 

in the cells, but that they became disorganized within a very few days. 
He attributed this to defective symbiotic relations between host and 
parasite. Miss Marryat (1907) showed that Puccinia glumarum when 
grown on a semi-immune host plant killed local areas of the host,, sent 
out but few haustoria, and never developed any but small or abortive 
pustules. 

It is a matter of common observation that in dealing either with 
cereals uncongenial to a given biologic form of Puccinia graminis or 
with varieties of wheat resistant to Puccinia graminis tritici flecks are 
often visible after inoculation, but no pustules, or only small ones, are 
produced. Examples of this are shown in Plate I, A and Plate VI, A. 
All degrees of this killing can be observed. The more readily the rust 
infects a plant the less likely are these dead areas to appear. When 
Puccinia graminis tritici is put on Minnesota No. 163 wheat, pustules 
are formed in great abundance, but the leaf tissues remain alive for a 
long time. When, on the other hand, resistant forms such as Kubanka 
2094 or Khapli are inoculated, areas of the leaf are killed outright ; and 
if pustules are formed at all they are very small. In extreme cases of 
incompatibility such as is found between Puccinia graminis avenae 
and wheat, the leaf area involved is usually so small that no indications 
of it can be seen with the unaided eye. 

Histological Details of Infection 
In order to determine the behavior of germ tubes in susceptible 
and nearly immune forms, leaves of Minnesota No. 163 and of Khapli 
were inoculated with Puccinia graminis tritici. Minnesota No. 163 was 
also inoculated with Puccinia graminis avenae. They were then placed 
in a pan of water under bell jars for 48 hours. Leaves were selected 
and killed every 24 hours, beginning with the first day. 

For killing, aceto-alcohol, medium chromo-acetic acid, and Flem- 
ming's weaker killing-fluid were used. The leaves were embedded 
in the usual manner and sectioned from five to ten microns thick. 
For staining, Haidenhain's iron-alum haematoxylin and orange G, 
Erlich's haematoxylin. Gram's stain and eosin, Delafield's haematoxylin 
used according to Durand's method, fuchsin and methyl green and the 
safranin, gentian violet, orange G. combination, used according to 
Harper's modification of Flemming's method, were used. The last 
named gave the best results. 

INFECTION OF MINNESOTA NO. 163 

At the end of 24 hours many of the spores have usually germ- 
inated, sending out long germ tubes, although some apparently 
germinate later. Two tubes may be sent out from the same spore, 
but usually one develops more vigorously than the other. The tube 
usually follows the epiderm closely ; swellings are often found above 



RUST-RESISTANT VARIETIES OF WHEAT 43 

the wall between two epidermal cells (Plate VII, 3). In these swell- 
ings, which appear very much like very young appressoria, the pro- 
toplasm often aggregates more densely than in the other parts of the 
tube. The germ tube may often follow the epiderm for considerable 
distances, sometimes for the length of 15 epidermal cells, before a 
definite appressorium is formed. Nearly always when a stoma is 
reached the tube forms a very definite swelling (Plate VII, 1) which 
constitutes an appressorium. The protoplasmic contents of practically 
the entire germ tube are concentrated in this appressorium. It dips 
down into the stomatal opening and a fine protoplasmic process is sent 
through to the substomatal space. Here a definite swelling takes 
place, forming the substomatal vesicle (Plate VII, 4 and 5). Appar- 
ently, in some cases, the vesicle develops no further. In the great 
majority of cdses, however, the protoplasm aggregates in it and 
infection-threads are sent out. Very often these infection-threads 
follow closely along under the epiderm cells and send small knoblike 
or sometimes flattened haustoria into the host cells. Branching then 
takes place among the cells of the leaf, many haustoria being sent out 
and the hyphae developing very rapidly. The threads may grow 
directly across the substomatal space and branch between the mesophyll 
cells (Plate VII, 6). This does not seem to be the usual method, 
however. Nuclear division takes place rapidly to keep pace with the 
growth of the hyphae. Frequently a number of nuclei are found in a 
single cell. The entire hypha often retains its protoplasm for a con- 
siderable length of time. Ordinarily the tip only remains densely 
protoplasmic while the rest of the hypha becomes much vacuolated. 
Very long hyphae are often found growing very vigorously but appar- 
ently not sending out haustoria. They seem to be in the nature of 
distributive filaments. The fungus does not seem to spread very far 
from the point of infection. When large areas of the leaf are involved, 
a number of points of entry can nearly always be found. On the 
fourth day an infected area is usually well filled with much branched 
hyphae. About this time wefts begin to be formed, the protoplasm 
aggregating in the tips of the hyphae. A dense mass of filaments is 
formed just beneath the epiderm and the epidermal cells are wedged 
apart. The large, upright fungus cells, which show the binucleate 
condition very clearly, begin to form uredospores. This is well under 
way on the fifth day and by the sixth or seventh day the epiderm has 
been completely ruptured while spores are being shed in great pro- 
fusion. It is interesting to note that the host cells, which are often 
half filled with large knoblike, filamentous or coiled haustoria, are 
usually still quite healthy at the time pustule formation begins. 

The whole appearance of both fungus and host during the first 
few days after infection indicates a fairly perfect relation between 



44 A STUDY IN CEREAL RUSTS 

the two. The fungus flourishes vigorously while for a considerable 
length of time the host cells, even in the infected area, are apparently 
quite healthy. In no case does there seem to be an extensive killing of 
host tissue. 

INFECTION OF KHAPLI 

Spore germination of course occurs normally. The germ tubes 
grow along the surface of the host epidefm cells in the same manner 
as do those on Minnesota No. 163. The formation of appressoria 
takes place in an entirely normal manner. The stimulus causing entry 
into the stomatal slit is present, the method of entrance being sub- 
stantially the same as in Minnesota No. 163. Apparently the vesicle 
sometimes fails to send out infection threads but merely remains 
directly beneath the stomatal slit and becomes vacuolated. It may send 
out numerous, short, club-shaped branches all of which soon become 
vacuolated and never send out any haustoria into the host cells. From 
the beginning of growth in the host it is easily discernible that the 
vigor of the hyphae is not nearly so great as is the case with those 
growing in Minnesota No. 163. There is a greater tendency for the 
tips of infection threads to round up, become vacuolated, and never 
develop further. 

Fairly successful infection, however, may take place. Infection 
threads may be sent out just under the epiderm or directly across the 
substomatal space (see Plate VIII, 3 and 5). Haustoria, attached to 
the hypha by delicate sterigmata, are sent into the cells and the hypha 
grows fairly well. Sometimes many incipient infection threads are 
formed from a single vesicle, only one developing (Plate VHI, 5). 
Shortly after infection threads are sent out the vesicle usually dies. 

The infection threads are not always successful in sending 
haustoria into the host cells. When the hypha comes into contact 
with the cells the protoplast of the latter often shrinks back from the 
wall, the nucleus shows definite signs of disintegration, the chloroplasts 
are apparently lost, and the entire cell dies. The hypha may die also, 
or it may grow and kill other cells. However, it usually eventually 
succumbs. Typical examples of this will be seen on Plate VIII, 6 and 
7. It will be noticed that at 6 the cell on which the hypha is abutting 
is apparently dead, the chloroplasts have disappeared, and the nucleus 
is disintegrating. At 7 this has taken place in only a part of the cell. 
It sometimes happens that one branch of a hypha is fairly successful 
while another may never develop to any extent at all. 

Whether or not the host cells are killed within a short time after 
the hyphae come in contact with them, infection does not appear to 
be normal. The hyphae may grow fairly well, but never as vigorously 
as in Minnesota No. 163. Haustoria mav be sent out in fair abundance 



RUST-RESISTANT VARIETIES OF WHEAT 45 

(Plate IX, 1, 2, and 4). The hyphal tips may branch very profusely 
but they become vaculoated very early in most cases and appear un- 
thrifty. It requires 8 or 9 days for the stage of infection to be 
reached on Khapli that is reached in 3 or 4 days in susceptible varieties. 
Short, thick, or rounded hyphal segments are quite common, those 
at the end of a branch often containing as many as 5 or 6 nuclei, some 
of which appear to be distintegrating. 

The tips of hyphae naturally die when a group of host cells among 
which they are growing is killed. However, they may disintegrate 
without having first killed host cells (Plate IX, 1, 2, 3, and 4). There 
may be many variations. The hyphae may not send out haustoria and 
die in consequence, or, even if they do send them into the cells, death 
may occur. Branches of hyphae which have sent haustoria into host 
cells frequently become vacuolated and gradually die, or the pro- 
toplasmic contents may change to granular, deep-staining masses. 
The whole appearance suggests fungous hyphae growing in an un- 
favorable nutrient solution. 

In about 11 or 12 days a distinct tendency toward the formation 
of hyphal wefts can be observed. These vary greatly both in size 
and position. They may be mere aggregations directly under the 
epiderm or deeper down in the tissues ; or they may begin to wedge 
the epiderm cells apart after the manner of young pustules. Often 
small, unruptured pustules are formed in which there is a fairly large 
number of abortive spores. The pustules may rupture the epiderm 
but they are always extremely small, and as a rule the spores are 
small. The average size of a large number of spores measured was 
29.69 X 20.68 yu, whereas the average size of the wheat rust spores 
which were used in making the inoculations was 35.38x21.39 i-i- 

After about 20 days practically all the host cells and a large 
number of the hyphae in an infected region are dead. Haustoria may 
be present in the host cells in fairly large numbers, but most of them 
are dead, their protoplasmic contents having broken up into granular, 
deep-staining masses. The nuclei of the host cells are often dis- 
integrating also. The infection by this time, and usually earlier, has 
completely run its course. Comparatively very few spores have been 
produced, and, under natural conditions, secondary infections would 
probably not occur to any extent. 

THE COURSE OF INFECTION IN OTHER RESISTANT FORMS 

Substantially the same sequence of events occurs in other resistant 
forms, such as Arnautka 288, Kubanka 2094, emmer 1522, einkorn 2433 
(sometimes), and in such cases as the infection of rye by barley rust. 
The differences seem to be in degree rather than in kind. In the cases 
of emmer and einkorn the killing of host cells is rarely found but 



46 A STUDY IN CEREAL RUSTS 

unsuccessful attempts to form pustules are often noticed. The 
sequence of events in Arnautka 288 and Kubanka 2094 is quite similar 
to that in Khapli. These are semi-immune forms in which the contest 
between host and parasite is somewhat prolonged. Fatalities occur on 
both sides but not in sufificient number to render infection absolutely 
unsuccessful. It is, therefore, essential to note the sequence in such 
extreme cases of almost total immunity as are furnished by wheat 
when inoculated with Puccinia graminis avenae. 

MINNESOTA NO. 163 INOCULATED WITH PUCCINIA GRAMINIS AVENAE 

It will be recalled that very rarely indeed does successful infection 
follow inoculation of wheat with Puccinia graminis avenae. Long 
germ tubes are sent out by spores germinating on the surface of the 
leaf. These follow the epiderm, dipping into depressions in an entirely 
normal manner. Appressoria are found, a small neck grows through 
the stomatal slit and the substomatal vesicle is formed. This vesicle 
sometimes sends out only very small knoblike branches which soon 
die, or branches may be sent out and very few hausteria produced. 

The vesicle often sends out many short, knoblike branches, 
appearing almost like elongated amoebae. These do not appear 
vigorous after a few days. The epiderm cells in the immediate vicinity 
appeared to be killed. If definite infection threads are sent out they 
never grow very long but kill two or three host cells and then stop 
growing. 

The difference between conditions in such a case as this and 
such a one as Khapli is apparently in degree only. Whereas in Khapli 
the fungus might develop to a certain extent, thus involving fairly 
large leaf areas, in such an extreme case of immunity as is presented 
by wheat to oat rust only a few host cells are involved and the contest 
between host and parasite is short and decisive, only a very few host 
cells being killed ; and the hyphae seldom develop sufficiently to give 
any external evidence that the germ tube has even entered. 

As to the fundamental causes for these facts, only speculation is 
possible. It would seem reasonable to suppose either that there was 
a lack of an attracting substance or the presence of a deleterious sub- 
stance. Massee's chemotaxis theory has already been mentioned. 
Chemotaxis or its absence would hardly explain the phenomena men- 
tioned, since the fungus succeeds in effecting an entrance into even the 
most immune forms. The evidence would rather seem to favor the 
view that the whole problem is one of toxins in host or parasite or, 
very probably, in both. In some cases the host is apparently hyper- 
susceptible, while further study may prove that there is in other cases 
a^ real resistance. Careful biochemical investigation alone can settle 
this question definitely. 



RUST-RESISTANT VARIETIES OF WHEAT 47 

Certainly, however, there can be no question of a certain antac^on- 
ism between host and parasite when one observes phenomena such as 
are ilkistratecl in Plate VIII, 6. This is in remarkably sharp contrast 
with the apparent congeniality exhibited in such cases as are shown 
at 6, 7, and 8 in Plate VII. Antagonism would seem to be explicable 
at present only by the toxin or enzyme theory. The recent work of 
Bolley (1908 and 1909), Pole Evans (1909 and 1911), McAlpine 
(1910), Freeman and Johnson (1911), and Biff en (1912) indicates 
clearly that immunity and resistance are concepts which, from the 
very nature of their variability and sometimes apparent capriciousness, 
must be cautiously discussed. At least one substance, commonly 
found in plants, has been found by Cook and Taubenhaus (1911, pp. 
40 and 43) to be toxic to certain rusts of the genus Uromyces. 

Whatever the immediate instruments governing congeniality or 
antagonism, the fundamental facts brought out quite clearly in the 
results described in the present investigation have a bearing on the 
practical and theoretical questions involved in the problem of pre- 
venting cereal rusts and the breeding of rust-resisting varieties. Ex- 
ternal morphology as pointed out by Ward (1902-1) for brome rusts, 
Salmon (1905-2) for mildews, and Biffen (1907) for yellow rust is 
also of very slight importance in the immunity of cereal varieties to 
stem rust. No observed facts in intimate histology, moreover, give 
any clue to resistance. In the absence of biochemical information 
concerning the activities of invading hyphae and invaded host tissues, 
actual performance alone can be depended upon as a safe criterion 
in the development of resistant forms or immune varieties. This is 
all the more true since Pole Evans (1911) has found that a hybrid 
wheat produced by crossing rust-immune and rust-susceptible wheats 
may rust quite badly and be capable of causing infection of the immune 
parent and a more severe attack of rust on the susceptible parent 
variety than rust from that variety itself will cause. The production 
of flecks and dead areas on an inoculated plant is a character of 
possible use in indicating at least a semi-immunity. 

There does not seem to be any obvious constant correlation be- 
tween immunity and other observable characters, as for instance 
drought-resistance. Although the immune varieties of wheat used 
in this investigation are drought-resistant, it is also a well-estab- 
lished fact that other drought-resistant wheats such as Kubanka 1516 
are very susceptible. Moreover, the antagonism exhibited by Minne- 
sota No. 163 wheat toward Puccinia graminis avenae and by rye 
toward Puccinia graminis hordei does not differ fundamentally from 
that exhibited by Khapli toward Puccinia graminis tritici and in the 
first two cases a correlation with drought-resistance is out of the 
question. 



48 A STUDY IN CEREAL RUSTS 

That the important scientific questions involved in the specializa- 
tion of biologic forms and that of rust-resistant varieties of wheat 
are essentially the same seems obvious. The same phenomena can be 
observed in both; there are various degrees of resistance and suscep- 
tibility in both and a thorough investigation with refined biochemical 
methods will probablv not only prove the similarity, but show the real 
reason for resistance and susceptibility. 

SUMMARY OF PART II 

1. In making inoculations in the greenhouse on wheats resistant 
to Puccinia graminis tritici it was found that only two, Khapli and 
Kubanka 2094, especially the former, possessed a very marked degree 
of real resistance, although a number of others were fairly resistant 
in the field. 

2. It was observed that the more resistant a form proved, the 
more pronounced was the tendency of the rust to kill small areas of 
the leaf. The pustules developed in these areas were always very 
small. 

3. The length of the incubation period of the rust is correlated 
to a certain extent with the degree of immunity, the most nearly 
immune forms, as a rule, having the longest incubation period. 

4. On the most resistant varieties, such as Khapli, the spores 
are often small in size and sometimes abortive. 

5. Infection secured on partially resistant varieties as a result 
of inoculations with aecidiospores and primarv uredospores proved 
only slightly more virulent than did that secured by means of inocula- 
tion with long-time uredospores. 

6. Drought-resistant durum wheats grown in very dry soil 
rusted more severely than those grown in soil with a higher moisture 
content. Minnesota No. 163 did not show much difference, the plants 
in wet soil being slightly more severely attacked. The conditions 
normal for the host plant are probably also the conditions under 
which the rust develops best. 

7. It was found that in general the absence or presence, in 
excessive amounts, of various nutrient substances, such as nitrogen 
and phosphorus salts, did not directly affect the immunity or suscepti- 
bility of wheats. Conditions favoring a normal development of the 
host were conducive to vigorous development of the rust. The action 
of fertilizers, either natural or artificial, is probably indirect. Tem- 
perature conditions and relative humidity of the atmosphere are prob- 
ably more important than soil conditions. 

8. The addition of copper sulfate, copper carbonate, and iron 
sulfate to nutrient media in which plants inoculated with rust were 
grown did not markedly diminish the amount of rust when they were 



RUST-RESISTANT VARIETIES OF WHEAT 49 

used in such concentration as to permit of the normal development 
of the host plants. 

9. A careful comparison of the sequence of infection in such 
a susceptible form as Minnesota No. 163 with that in such an immune 
form as Khapli reveals the fact that the fung-us i;ains entrance in 
the same manner in both cases. The rust mycelium is able to grow 
luxuriantly in Minnesota No. 163 and produce spores in .c^reat abun- 
dance. In Khapli, however, it does not thrive. The reason seems to 
be a physiological incompatibility as evidenced by the killing of host 
cells by the fungus and the more or less sudden death of the fungus 
itself. Infection may occur and pustules may be developed, but it is 
evident that the fungus is not in a congenial environment. The 
conditions seem to be essentially similar when examination is made of 
a cereal almost completely immune to a biologic form, such as Min- 
nesota No. 163, inoculated with Puccinia graminis avenae. Here, 
however, the host cells and rust hyphae are killed earlier and the leaf 
area involved is consequently smaller. This, however, requires further 
study. 

10. The question as to the immediate instruments of immunity 
can probably only be answered by means of biochemical investigations. 
In the meantime, morphological and histological characters being 
clearly of minor importance in determining immunity, only the per- 
formance of a supposedly resistant variety under varying conditions 
can be depended on for a criterion of its value in this respect. 

ACKNOWLEDGMENTS 

The writer takes pleasure in making acknowledgment to E. C. 
Johnson for suggestions and material, and especially to Dr. E. M. 
Freeman, under whom the work was done, for many suggestions and 
much criticism during the progress of the work. 



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52 A STUDY IN CERIAL RUSTS 

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sitischer Pilze. Hedw. 33: 362. 1894. 

Eine Bemerkung zu E. Fischer's erfolgreichen Infectionen einiger 

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54 A STUDY IN CEREAL RUSTS 

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EXPLANATION OF PLATES 

The drawings were all made with the aid of the camera lucida. The Zeiss 
3 mm. N. A. 1.3 homogeneous oil immersion and compensating ocular No. 4 
were used for all except Figures 8 and 10, Plate IX, where compensating ocular 
No. 6 was substituted. 

Plate I A. Rye after inoculation with Puccinia graminis hordet, showing dead 
leaf areas and a very few minute pustules. 

B. Normal but rather light infection on rye by P. graminis secalis, showing 
absence of dead areas. 

Plate II A. Einkorn 2433 inoculated with P. graminis originally obtained from 
wheat and confined to Einkorn for twenty-five successive generations. 
B. Einkorn 2433 inoculated with P. graminis from wheat. 

Plate III A. Wheat, Minnesota No. 163, inoculated with rust which had been 
confined to Einkorn for twenty-five generations. 

B. Normal infection of wheat following inoculation with P. graminis tritici 
from wheat. 

Plate IV A. Kubanka 1516 grown in very wet soil after inoculation with 
P. graminis. 
B. Kubanka grown in very dry soil after inoculation. 

Plate V A. lumillo 1736 grown in very wet soil after inoculation with P. 
graminis tritici. 

B. The same variety inoculated under same condition but grown in very 
dry soil. 

Plate VI A. Khapli, showing characteristic infection following heavy inoculation 
with P. graminis. 
B. Normal infection on Minnesota No. 163. 

Plate VII. Infection of Minnesota No. 163 wheat by P. graminis tritici. 
1, 2 and 3 — 48 hours after inoculation. 
4 and 5 — 72 hours after inoculation. 
6 to 9 — 5 days after inoculation. 

1. Surface view showing appressorium forming over a stoma. 

2. Appressorium being formed directly, without germ tube development. 

3. Germ tube apparently passing over stoma and forming a swelling— an 

unusual occurrence. 

4. Part of a germ tube, appressorium, and neck connecting the appressorium 
with the substomatal vesicle which has been cut at one side. 

5. Substomatal vesicle, cut at one side, beginning to branch. 

6. Infection thread growing from vesicle directly across substomatal space ; 

remains of appressorium outside. (Section slightly torn.) 

7. Part of an infection thread showing haustorium in epidermal cell. 
8 and 9. Later stages showing development of long hyphae. 

Plate VIII. Infection of Khapli with P. graminis tritici. 
1 and 2 — 72 hours after inoculation. 
3 and 4 — 6 days after inoculation. 
5, 6 and 7 — 8 days after inoculation. 

55 



56 A STUDY IN CEREAL RUSTS 

1. Part of a germ tube and an appressorium. 

2. Germ tube, appressorium, and substomatal vesicle into which the nuclei 
have passed. 

3. Infection thread, with a few short branches, killing the host cell. The 
protoplast has shrunk and the nucleus is disintegrating. 

4. Infection thread in contact with a cell which it is apparently killing. 

5. Substomatal vesicle and a number of somewhat abortive infection 
threads. In the epidermal cell on the left two haustoria, deeply stained, 
and possibly dead. Below, fairly successful infection. 

6. Empty appressorium and vesicle. On the left infection threads, one 

of which has sent a haustorium into an epidermal cell. 

7. Infection threads growing toward leaf tissues. 

Plate IX. Same as Plate VIII, except Figs. 6 to 10, which are of Arnautka. 
1 to 5 — 10 days after inoculation. 
6 to 10 — 23 days after inoculation. 

1. Short hyphal segment containing four nuclei. Haustorium in epidermal 
cell. Hyphae sent out from segment disintegrating. 

2. Disintegrating hypha with haustorium in host cell. 

3. Long hyphae, the tips of some becoming much vacuolated and apparently 
dying. 

4. Typical appearance of hyphae under epiderm, showing haustoria and 
some dying hyphal tips. 

5. Hyphae deeper down in leaf tissues showing tendency to aggregate. 

6. Dead host cells and practically dead hyphae. 

7. Hyphae typical of those in subepidermal wefts showing knoblike branches 
which are often quite characteristic. 

8. Small, partly ruptured pustule. 

9. Single uredospore. 

10. Subepidermal weft showing unsuccessful attempt at pustule formation 
and a number of abortive spores. 



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